U.S. patent application number 16/246321 was filed with the patent office on 2019-07-18 for nucleic acid constructs, plants with increased tuber yield, and methods for increasing tuber yield in a plant.
The applicant listed for this patent is India Institute of Science Education and Research (IISER), Iowa State University Research Foundation, Inc.. Invention is credited to Anjan K. Banerjee, David J. Hannapel.
Application Number | 20190218565 16/246321 |
Document ID | / |
Family ID | 67212739 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190218565 |
Kind Code |
A1 |
Hannapel; David J. ; et
al. |
July 18, 2019 |
NUCLEIC ACID CONSTRUCTS, PLANTS WITH INCREASED TUBER YIELD, AND
METHODS FOR INCREASING TUBER YIELD IN A PLANT
Abstract
The present invention relates to a nucleic acid construct. The
nucleic acid construct includes a nucleic acid molecule configured
to silence or reduce the expression of StBEL11 and/or StBEL29 and
variants thereof, a 5' DNA promoter sequence, and a 3' terminator
sequence, where the first nucleic acid molecule, the promoter
sequence, and the terminator sequence are operatively coupled to
permit transcription of the first nucleic acid molecule. The
present invention is also directed to vectors, host cells,
transgenic plants, and transgenic plant seeds, as well as
non-transgenic mutant plants, and methods for altering tuber yield
in a plant.
Inventors: |
Hannapel; David J.; (Ames,
IA) ; Banerjee; Anjan K.; (Pune, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Iowa State University Research Foundation, Inc.
India Institute of Science Education and Research (IISER) |
Ames
Pune |
IA |
US
IN |
|
|
Family ID: |
67212739 |
Appl. No.: |
16/246321 |
Filed: |
January 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62616565 |
Jan 12, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8234 20130101;
C12N 15/8226 20130101; C12N 15/8261 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Goverment Interests
[0002] This invention was made with government support under grant
number DBI0820659 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A nucleic acid construct comprising: a first nucleic acid
molecule comprising a nucleotide sequence configured to silence or
reduce expression of StBEL11 and variants thereof; a 5' DNA
promoter sequence; and a 3' terminator sequence, wherein the first
nucleic acid molecule, the promoter sequence, and the terminator
sequence are operatively coupled to permit transcription of the
first nucleic acid molecule.
2. The nucleic acid construct according to claim 1, wherein the
nucleic acid construct comprises DNA heterologous to the first
nucleic acid molecule.
3. The nucleic acid construct according to claim 2, wherein the DNA
heterologous to the first nucleic acid molecule is the 5' DNA
promoter sequence.
4. The nucleic acid construct according to claim 1, wherein the
first nucleic acid molecule is SEQ ID NO:3 or a nucleotide sequence
that is at least 95% identical to the nucleotide sequence of SEQ ID
NO:3.
5. The nucleic acid construct according to claim 1 further
comprising: a second nucleic acid molecule comprising a nucleotide
sequence configured to silence or reduce expression of StBEL29 and
variants thereof.
6. The nucleic acid construct according to claim 5, wherein the
second nucleic acid molecule is SEQ ID NO:6 or a nucleotide
sequence that is at least 95% identical to the nucleotide sequence
of SEQ ID NO:6.
7. The nucleic acid construct according to claim 1 further
comprising: a further nucleic acid molecule comprising a nucleotide
sequence configured to enhance the expression of StBEL5 and
variants thereof.
8. The nucleic acid construct according to claim 7, wherein the
further nucleic acid molecule comprises a nucleotide sequence of
SEQ ID NO:8 or a nucleic acid molecule that is at least 99%
identical to the nucleotide sequence of SEQ ID NO:8.
9. A nucleic acid construct comprising: a first nucleic acid
molecule comprising a nucleotide sequence configured to silence or
reduce expression of StBEL29 and variants thereof; a 5' DNA
promoter sequence; and a 3' terminator sequence, wherein the first
nucleic acid molecule, the promoter sequence, and the terminator
sequence are operatively coupled to permit transcription of the
first nucleic acid molecule.
10. The nucleic acid construct according to claim 9, wherein the
nucleic acid construct comprises DNA heterologous to the first
nucleic acid molecule.
11. The nucleic acid construct according to claim 10, wherein the
DNA heterologous to the first nucleic acid molecule is the 5' DNA
promoter sequence.
12. The nucleic acid construct according to claim 9, wherein the
first nucleic acid molecule comprises SEQ ID NO:6 or a nucleotide
sequence that is at least 95% identical to the nucleotide sequence
of SEQ ID NO:6.
13. The nucleic acid construct according to claim 9 further
comprising: a further nucleic acid molecule comprising a nucleotide
sequence configured to enhance the expression of StBEL5 and
variants thereof.
14. The nucleic acid construct according to claim 13, wherein the
further nucleic acid molecule comprises a nucleotide sequence of
SEQ ID NO:8 or a nucleic acid molecule that is at least 99%
identical to the nucleotide sequence of SEQ ID NO:8.
15. The nucleic acid construct according to claim 1, wherein the
promoter sequence is one or more of the following: a native,
constitutive, inducible, developmentally-regulated,
organelle-specific, tissue-specific, cell-specific, seed specific,
and germination-specific promoter.
16. An expression vector comprising the nucleic acid construct
according to claim 1.
17. A host cell transformed with the nucleic acid construct
according to claim 1.
18. The host cell according to claim 17, wherein the host cell is a
bacterial cell or a plant cell.
19. A transgenic plant seed transformed with the nucleic acid
construct according to claim 1.
20. A transgenic plant transformed with the nucleic acid construct
according to claim 1, wherein the plant has increased tuber yield
compared to a plant not transformed with the nucleic acid
construct.
21. The transgenic plant of claim 20, wherein tuber yield is
increased at least 20% compared to a plant not transformed with the
nucleic acid construct.
22. The transgenic plant of claim 20, wherein overall shoot fresh
weight is at least 80% compared to a plant not transformed with the
nucleic acid construct.
23. The transgenic plant of claim 20, wherein the plant comprises
an expression level of StBEL11 less than 60% compared to a plant
not transformed with the nucleic acid construct.
24. The transgenic plant of claim 20, wherein the plant comprises
an expression level of StBEL29 less than 60% compared to a plant
not transformed with the nucleic acid construct.
25. The transgenic plant of claim 20, wherein the plant comprises
an expression level of StBEL5 greater than 20% compared to a plant
not transformed with the nucleic acid construct.
26. The transgenic plant according to claim 20, wherein the plant
is selected from the group consisting of potato (Solanum
tuberosum), dahlia, caladium, Jerusalem artichoke (Helianthus
tuberosus), yam (Dioscorea alta), sweet potato (Impomoea batatus),
cassaya (Manihot esculenta), tuberous begonia, cyclamen, other
solarium species (e.g., wild potato), sugar beet (Beta vulgaris),
carrot (Daucus carota), and radish (Raphanus sativus).
27. The transgenic plant according to claim 20, wherein the plant
is selected from the group consisting of Solanum tuberosum spp.
andigena and Solanum tuberosum spp. tuberosum.
28. A transgenic cell of the plant according to claim 20.
29. A transgenic plant seed produced from the plant according to
claim 20.
30. A method of increasing tuber yield in a plant, said method
comprising: providing a transgenic plant or plant seed comprising a
nucleic acid construct comprising one or more nucleic acid
molecules configured to reduce or silence expression of (i) StBEL11
and variants thereof, (ii) StBEL29 and variants thereof, or (iii)
both (i) and (ii); and growing the transgenic plant or plant grown
from the transgenic plant seed under conditions effective to
express the one or more nucleic acid molecules in said transgenic
plant or said plant grown from the transgenic plant seed.
31. The method according to claim 30, wherein a transgenic plant is
provided.
32. The method according to claim 30, wherein a transgenic plant
seed is provided.
33. The method according to claim 30, wherein said providing
comprises transforming a non-transgenic plant or a non-transgenic
plant seed with the nucleic acid construct to yield said transgenic
plant or plant seed.
34. A potato plant comprising one or more mutations in one or both
of StBEL11 and StBEL29, wherein said potato plant has increased
tuber yield compared to the tuber yield of a wild type potato
plant.
35. The potato plant of claim 34, wherein the potato plant
comprises a reduced expression level of StBEL11 compared to a wild
type potato plant.
36. The potato plant of claim 35, wherein the potato plant
comprises an expression level of StBEL11 less than 60% compared to
a wild type potato plant.
37. The potato plant of claim 35, wherein the expression level of
StBEL11 is measured by quantifying accumulation levels of StBEL11
in leaves of a young tissue culture plant using RT-qPCR.
38. The potato plant of claim 34, wherein the potato plant
comprises a reduced expression level of StBEL29 compared to a wild
type potato plant.
39. The potato plant of claim 38, wherein the potato plant
comprises an expression level of StBEL29 less than 60% compared to
a wild type potato plant.
40. The potato plant of claim 38, wherein the expression level of
StBEL29 is measured by quantifying accumulation levels of StBEL11
in leaves of a young tissue culture plant using RT-qPCR.
41. The potato plant of claim 34, wherein the potato plant
comprises a tuber yield at least 20% greater than a wild type
potato plant.
42. A potato plant according to claim 34, wherein the potato plant
comprises an increased expression level of StBEL5 compared to a
wild type potato plant.
43. The potato plant of claim 42 further comprising: one or more
mutations in StBEL5.
44. The potato plant of claim 42, wherein the potato plant
comprises an expression level of StBEL5 greater than 20% compared
to a wild type potato plant.
45. The potato plant according to claim 34, wherein overall shoot
fresh weight is at least 90% compared to a plant not transformed
with the nucleic acid construct.
46. Potato seed from the potato plant of claim 34.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/616,565, filed Jan. 12, 2018, which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to nucleic acid constructs,
transgenic and non-transgenic plants with increased tuber yield,
and methods for increasing tuber yield in a plant.
BACKGROUND OF THE INVENTION
[0004] There are thirteen functional BELL-like genes in potato
(Solanum tuberosum L.) that encode for a family of transcription
factors ("TF") ubiquitous in the plant kingdom. These BEL1 TFs work
in tandem with KNOTTED1-types to regulate the expression of
numerous target genes involved in hormone metabolism and growth
processes. One of the StBELs, StBEL5, functions as a long-distance
mRNA signal that is transcribed in leaves and moves into roots and
stolons to stimulate growth. The two most closely related StBELs to
StBEL5 are StBEL11 and StBEL29. Together, these three genes make up
more than 70% of all StBEL transcripts present throughout the
potato plant. They share a number of common features, suggesting
they may be co-functional in tuber development. Upstream sequence
driving GUS expression in transgenic potato lines demonstrated that
both StBEL11 and StBEL29 promoter activity is robust in leaf veins,
petioles, stems, and vascular tissues and induced by short-days in
leaves and stolons. Steady-state levels of their mRNAs were also
enhanced by short-day conditions in specific organs.
[0005] Numerous plant developmental processes are known to be
regulated by the three amino acid loop extension ("TALE") family of
proteins. The TALE family includes BEL1-like ("BELL") and
KNOTTED1-like homeobox ("KNOX") transcription factors (TFs)
(Burglin et al., "Analysis of TALE Superclass Homeobox Genes (MEIS,
PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between
Plants and Animals," Nucleic Acids Research 25:4173-4180 (1997)).
Both BELL and KNOX proteins have characteristic
proline-tyrosine-proline (P--Y--P) residues between helix I and II
of their homeodomain (Passner et al., "Structure of DNA-Bound
Ultrabithorax-Extradenticle Homeodomain Complex," Nature 397:714-19
(1999)). BELL proteins have a DNA-binding homeodomain, a conserved
SKY box, a KNOX protein-interacting BELL domain, and a conserved
VSLTLGL motif. Both TALE types are ubiquitous in plants, and during
evolution of a number of gene family members have increased in
correlation with enhanced complexity. Red and green algal species
have only one or two KNOX and BELL genes, whereas land plant
genomes contain several genes of both (Mukherjee et al., "A
Comprehensive Classification and Evolutionary Analysis of Plant
Homeobox Genes," Mol. Biol. Evol. 26:2775-94 (2009)).
[0006] The BEL1 TF from Arabidopsis, the first BEL1 protein
discovered, functions in ovule and integument development (Ray et
al., "Arabidopsis Floral Homeotic Gene BELL (BELT) Controls Ovule
Development Through Negative Regulation of AGAMOUS Gene," Proc.
Nat'l. Acad. Sci. U.S.A. 97:5761-65 (1994); Reiser et al., "The
BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern
Formation in the Arabidopsis Ovule Primordium," Cell 83:735-42
(1995)). BEL1 TFs regulate numerous processes in plants such as
development of the shoot apical meristem ("SAM") (Byrne et al.,
"Phyllotactic Pattern and Stem Cell Fate are Determined by the
Arabidopsis Homeobox Gene BELLRINGER," Development 130:3941-50
(2003); Rutjens et al., "Shoot Apical Meristem Function in
Arabidopsis Requires the Combined Activities of Three BEL1-Like
Homeodomain Proteins," Plant J. 58:641-54 (2009)), control of
inflorescence architecture (Bhatt et al., "VAAMANA-a BEL1-Like
Homeodomain Protein, Interacts With KNOX Proteins BP and STM and
Regulates Inflorescence Stem Growth in Arabidopsis," Gene
328:103-11 (2004); Ragni et al., "Interaction of KNAT6 and KNAT2
with BREVIPEDICELLUS and PENNYWISE in Arabidopsis Inflorescences,"
Plant Cell 20:888-900 (2008)), leaf patterning (Kumar et al., "The
Arabidopsis BEL1-LIKE HOMEODOMAIN Proteins SAW1 and SAW2 Act
Redundantly to Regulate KNOX Expression Spatially in Leaf Margins,"
Plant Cell 19:2719-35 (2007)), the high-irradiance response of
phytochrome A (Staneloni et al., "Bell-Like Homeodomain Selectively
Regulates the High-Irradiance Response of Phytochrome A," Proc.
Nat'l. Acad. Sci. U.S.A. 106:13624-29 (2009)), regulation of
tuberization (Chen et al., "Interacting Transcription Factors From
the Three Amino Acid Loop Extension Superclass Regulate Tuber
Formation," Plant Physiol. 132:1391-1404 (2003); Banerjee et al.,
"Efficient Production of Transgenic Potato (S. tuberosum L. ssp.
andigena) Plants via Agrobacterium Tumefaciens-Mediated
Transformation," Plant Sci. 170:732-38 (2006)) and fruit
development (Dong et al., "MDH1: An Apple Homeobox Gene Belonging
to the BEL1 Family," Plant Mol. Biol. 42:623-33 (2000)).
[0007] The BEL1-KNOX tandem complex functions as a transcriptional
switch that regulates various developmental pathways in plants (Hay
& Tsiantis, "KNOX Genes: Versatile Regulators of Plant
Development and Diversity," Development 137:3153-65 (2010)). For
example, the BELLRINGER and shoot meristemless (STM) heterodimer in
Arabidopsis maintains SAM and inflorescence patterning (Byrne et
al., "Phyllotactic Pattern and Stem Cell Fate are Determined by the
Arabidopsis Homeobox Gene BELLRINGER," Development 130:3941-50
(2003); Roeder et al., "The Role of the REPLUMLESS Homeodomain
Protein in Patterning the Arabidopsis Fruit," Curr. Biol.
13:1630-35 (2003)). The BLH1 and KNAT3 heterodimer regulates seed
germination and early seedling development in Arabidopsis (Kim et
al., "BLH1 and KNAT3 Modulate ABA Responses During Germination and
Early Seedling Development in Arabidopsis," Plant J. 75:755-66
(2013)). In potato, the BEL1-KNOX interaction is functional in
regulating the tuberization process and root growth (Chen et al.,
"Interacting Transcription Factors From the Three Amino Acid Loop
Extension Superclass Regulate Tuber Formation," Plant Physiol.
132:1391-1404 (2003); Lin et al., "The Impact of the Long-Distance
Transport of a BEL1-Like mRNA on Development," Plant Physiol.
161:760-72 (2013)). Specifically, the StBEL5-POTH1 heterodimer
appears to regulate tuber formation in potato by regulating
transcription of target genes that are involved in hormone
metabolism. These include genes involved in auxin, cytokinin, and
gibberellic acid ("GA") synthesis and activity (Chen et al.,
"Interacting Transcription Factors From the Three Amino Acid Loop
Extension Superclass Regulate Tuber Formation," Plant Physiol.
132:1391-1404 (2003); Hannapel et al., "Phloem-Mobile Messenger
RNAs and Root Development," Front. Plant. Sci. 4:257 (2013); Lin et
al., "The Impact of the Long-Distance Transport of a BEL1-Like mRNA
on Development," Plant Physiol. 161:760-72 (2013)). GA levels are
reduced in newly formed tubers, whereas cytokinin and auxin levels
are enhanced (Machackova et al., "Photo-Periodic Control of Growth,
Development and Phytohormone Balance in Solanum tuberosum,"
Physiol. Plant 102:272-78 (1998); Xu et al., "The Role of
Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato
Tuber Formation In Vitro," Plant Physiol. 117:575-84 (1998);
Bou-Torrent et al., "Gibberellin A1 Metabolism Contributes to the
Control of Photo-Period-Mediated Tuberization in Potato," PLoS One
6:e24458 (2011); Roumeliotis et al., "A Crosstalk of Auxin and GA
During Tuber Development," Plant Signal. Behav. 7:1360-63 (2012);
Abelenda et al., "Flowering and Tuberization: A Tale of Two
Nightshades," Trends Plant Sci. 19:115-22 (2014)). Included among
these target genes are StPIN1, -2, and -4; several AUX/IAA types;
StLONELYGUY1, -2, and -3; StGA2 oxidase1; and StGA20 oxidase1 (Chen
et al., "Interacting Transcription Factors From the Three Amino
Acid Loop Extension Superclass Regulate Tuber Formation," Plant
Physiol. 132:1391-1404 (2003); Hannapel et al., "Phloem-Mobile
Messenger RNAs and Root Development," Front. Plant. Sci. 4:257
(2013); Lin et al., "The Impact of the Long-Distance Transport of a
BEL1-Like mRNA on Development," Plant Physiol. 161:760-72 (2013);
Sharma et al., "Targets of the StBEL5 Transcription Factor Include
the FT Ortholog StSP6A," Plant Physiol. 170:310-24 (2016)). The
StBEL5-POTH1 complex binds to tandem TTGAC motifs present in
upstream sequence of these target genes. As an example, StGA2ox1,
which is strongly induced during early tuber formation (Kloosterman
et al., "StGA2ox1 is Induced Prior to Stolon Swelling and Controls
GA Levels During Potato Tuber Development," Plant J. 52:362-73
(2007)), contains four sets of tandem TTGAC elements in its
upstream sequence and two in its first intron (Lin et al., "The
Impact of the Long-Distance Transport of a BEL1-Like mRNA on
Development," Plant Physiol. 161:760-72 (2013)). Each of the two
TFs binds to one of the TTGAC core motifs and both are required to
affect transcription (Chen et al., "The Tandem Complex of BEL and
KNOX Partners is Required for Transcriptional Repression of
ga20ox1," Plant J. 38:276-84 (2004)).
[0008] Both StBEL5 and POTH1 transcripts were detected in phloem
cells (Banerj ee et al., "Dynamics of a Mobile RNA of Potato
Involved in a Long-Distance Signaling Pathway," Plant Cell
18:3443-57 (2006); Yu et al., "Tissue Integrity and RNA Quality of
Laser Microdissected Phloem of Potato," Planta 226:797-803 (2007);
Lin et al., "Transcriptional Analysis of Phloem-Associated Cells of
Potato," BMC Genom. 16:665 (2015)). Both RNAs have been proposed to
act as long-distance signals (Banerjee et al., "Dynamics of a
Mobile RNA of Potato Involved in a Long-Distance Signaling
Pathway," Plant Cell 18:3443-57 (2006); Mahajan et al., "The mRNA
of a Knotted1-Like Transcription Factor of Potato is Phloem
Mobile," Plant Mol. Biol. 79:595-608 (2012)) and move freely
throughout the plant with enhanced movement of StBEL5 into stolons
under short-days (Banerjee et al., "Dynamics of a Mobile RNA of
Potato Involved in a Long-Distance Signaling Pathway," Plant Cell
18:3443-57 (2006)). Overexpression, movement, and accumulation of
StBEL5 RNA have been consistently associated with increased
earliness and enhanced tuber yields even under non-inductive
long-day conditions (Chen et al., "Interacting Transcription
Factors From the Three Amino Acid Loop Extension Superclass
Regulate Tuber Formation," Plant Physiol. 132:1391-1404 (2003);
Banerjee et al., "Untranslated Regions of a Mobile Transcript
Mediate RNA Metabolism," Plant Physiol. 151:1831-43 (2009)).
Movement of its mRNA to stolon tips in over-expressing plants is
facilitated by the presence of the untranslated regions of its RNA
(Banerjee et al., "Dynamics of a Mobile RNA of Potato Involved in a
Long-Distance Signaling Pathway," Plant Cell 18:3443-57 (2006);
Banerjee et al., "Untranslated Regions of a Mobile Transcript
Mediate RNA Metabolism," Plant Physiol. 151:1831-43 (2009)). RNA
binding proteins that bind to sequences in the 3' untranslated
region (UTR) of its transcript facilitate localized StBEL5 movement
and enhance tuberization. These RNA-binding proteins are induced by
short-day (SD) conditions (Cho et al., "Polypyrimidine
Tract-Binding Proteins of Potato Mediate Tuberization Through an
Interaction With StBEL5 RNA," J Expt. Bot. 66:6835-47 (2015)).
[0009] Other tuberization signals like the FT-ortholog StSP6A in
potato also accumulate in stolons of plants grown under SD (Navarro
et al., "Control of Flowering and Storage Organ Formation in Potato
by FLOWERING LOCUS T," Nature 478:119-22 (2011); Gonzalez-Schain et
al., "Potato CONSTANS is Involved in Photoperiodic Tuberization in
a Graft-Transmissible Manner," Plant J. 70:678-90 (2012)). The
microRNA, miR172, promotes tuber formation and accumulates in
stolons at the onset of tuberization (Martin et al.,
"Graft-Transmissible Induction of Potato Tuberization by the
MicroRNA miR172," Development 136:2873-81 (2009)). Moreover,
Bhogale et al., "MicroRNA156: A Potential Graft-Transmissible
MicroRNA That Modulates Plant Architecture and Tuberization in
Solanum tuberosum ssp. andigena," Plant Physiol. 164:1011-27 (2014)
suggested that miR156 acts as a phloem-mobile signal and regulates
aerial tuber formation in potato.
[0010] Recent work on transcription profiling of StBEL5 suggests
that it is positioned upstream of the regulatory network that
controls the onset of tuber formation (Sharma et al., "Targets of
the StBEL5 Transcription Factor Include the FT Ortholog StSP6A,"
Plant Physiol. 170:310-24 (2016)). Signaling targets of StBEL5
include the gene for earliness, StCDF1 (Kloosterman et al.,
"Naturally Occurring Allele Diversity Allows Potato Cultivation in
Northern Latitudes," Nature 495:246-50 (2013)), and the tuber
signal StSP6A (Navarro et al., "Control of Flowering and Storage
Organ Formation in Potato by FLOWERING LOCUS T," Nature 478:119-22
(2011)). Through its transcriptional activity in conjunction with
its KNOX partner, StBEL5 front-loads the tuber signals, StSP6A and
StCDF1, in the leaf and then follows this with a doubling-down of
the two key tuber signals, StSP6A and StBEL5, in stolons during the
onset of tuber formation. Auto-regulation of its own gene is also
occurring in the stolons. Site mutagenesis in tandem TTGAC motifs
(specific for the StBEL5/KNOX complex) located in the upstream
sequence of both StBEL5 and StSP6A suppressed the SD-induced
activity of their promoters in young tubers (Lin et al., "The
Impact of the Long-Distance Transport of a BEL1-Like mRNA on
Development," Plant Physiol. 161:760-72 (2013); Sharma et al.,
"Targets of the StBEL5 Transcription Factor Include the FT Ortholog
StSP6A," Plant Physiol. 170:310-24 (2016)). Suppression of StBEL5
activity repressed the accumulation of RNA for StSP6A, whereas
induction of StBEL5 had the opposite effect (Sharma et al.,
"Targets of the StBEL5 Transcription Factor Include the FT Ortholog
StSP6A," Plant Physiol. 170:310-24 (2016)).
[0011] Thirteen BEL1-like genes have been identified in the potato
genome and were organized into five clades based on amino-acid
sequence (Sharma et al., "The BEL1-Like Family of Transcription
Factors in Potato," J. Expt. Bot. 65:709-23 (2014)). StBEL5, -11,
and -29 are phylogenetically related and exhibit very close
sequence matches within their conserved domains. In addition, the
transcripts of these three StBEL genes make up more than 70% of the
total transcripts in the StBEL family (Xu et al., "Genome Sequence
and Analysis of the Tuber Crop Potato," Nature 475:189-95 (2011);
Sharma et al., "The BEL1-Like Family of Transcription Factors in
Potato," J. Expt. Bot. 65:709-23 (2014)). All three are present in
phloem cells and exhibit very high transcript levels in petioles, a
key organ for transporting RNAs into the stem. Similar to StBEL5,
these data suggest that StBEL11 and -29 might function as
phloem-mobile developmental signals. Although recent results have
demonstrated the dramatic effect that suppression of StBEL5 RNA had
on tuberization (Sharma et al., "Targets of the StBEL5
Transcription Factor Include the FT Ortholog StSP6A," Plant
Physiol. 170:310-24 (2016)), it is conceivable that StBEL11 and -29
are also involved in some aspect of tuber formation. Other than
StBEL5, very little is known about the functional roles played by
other members of the StBEL family.
[0012] The present invention is directed to overcoming deficiencies
in the art.
SUMMARY OF THE INVENTION
[0013] One aspect of the present invention is directed to a nucleic
acid construct comprising a first nucleic acid molecule comprising
a nucleotide sequence configured to silence or reduce expression of
StBEL11 and variants thereof; a 5' DNA promoter sequence; and a 3'
terminator sequence, where the first nucleic acid molecule, the
promoter sequence, and the terminator sequence are operatively
coupled to permit transcription of the first nucleic acid
molecule.
[0014] Another aspect of the present invention is directed to a
nucleic acid construct comprising a first nucleic acid molecule
comprising a nucleotide sequence configured to silence or reduce
expression of StBEL29 and variants thereof; a 5' DNA promoter
sequence; and a 3' terminator sequence, where the first nucleic
acid molecule, the promoter sequence, and the terminator sequence
are operatively coupled to permit transcription of the first
nucleic acid molecule.
[0015] A further aspect of the present invention is directed to an
expression vector comprising a nucleic acid construct of the
present invention.
[0016] Another aspect of the present invention is directed to a
host cell transformed with a nucleic acid construct of the present
invention.
[0017] A further aspect of the present invention is directed to a
transgenic plant seed transformed with a nucleic acid construct of
the present invention.
[0018] Another aspect of the present invention is directed to a
transgenic plant transformed with a nucleic acid construct of the
present invention, where the plant has increased tuber yield
compared to a plant not transformed with the nucleic acid
construct.
[0019] A further aspect of the present invention relates to a
transgenic cell of a plant of the present invention.
[0020] Another aspect of the present invention relates to a
transgenic plant seed produced from a plant of the present
invention.
[0021] A further aspect of the present invention is directed to a
method of increasing tuber yield in a plant. This method involves
providing a transgenic plant or plant seed comprising a nucleic
acid construct comprising one or more nucleic acid molecules
configured to reduce or silence expression of (i) StBEL11 and
variants thereof, (ii) StBEL29 and variants thereof, or (iii) both
(i) and (ii); and growing the transgenic plant or plant grown from
the transgenic plant seed under conditions effective to express the
one or more nucleic acid molecules in said transgenic plant or said
plant grown from the transgenic plant seed.
[0022] Another aspect of the present invention is directed to a
potato plant comprising one or more mutations in one or both of
StBEL11 and StBEL29, where the potato plant has increased tuber
yield compared to the tuber yield of a wild type potato plant.
[0023] A further aspect of the present invention relates to potato
seed from the potato plant comprising one or more mutations in one
or both of StBEL11 and StBEL29 of the present invention.
[0024] Using a transgenic approach and heterografting experiments,
it is shown herein that both StBEL11 and StBEL29 inhibit growth in
correlation with the long distance transport of their mRNAs from
leaves to roots and stolons, whereas suppression lines of these two
RNAs exhibited enhanced tuber yields. These results indicate that
the RNAs of StBEL11 and StBEL29 are phloem-mobile and function
antagonistically to the growth-promoting characteristics of StBEL5.
Both these RNAs appear to inhibit growth in tubers by repressing
the activity of target genes of StBEL5.
[0025] As is demonstrated herein, RNAs of StBEL11 and StBEL29 are
phloem-mobile and function antagonistically to the growth-promoting
characteristics of StBEL5 in potato. Both these RNAs appear to
inhibit tuber growth by repressing the activity of target genes of
StBEL5 in potato. Moreover, upstream sequence driving GUS
expression in transgenic potato lines demonstrated that both
StBEL11 and StBEL29 promoter activity is robust in leaf veins,
petioles, stems, and vascular tissues and induced by short days in
leaves and stolons. Steady-state levels of their mRNAs were also
enhanced by short-day conditions in selective organs.
[0026] To expand the understanding of long-distance signaling and
to determine if they have any relationship with plant growth and
tuberization, expression profiles and functional analyses of
StBEL11 and StBEL29, which are closely related to StBEL5, were
undertaken as part of the present invention. Similar to StBEL5, the
results described herein suggest that the RNAs of StBEL11 and
StBEL29 function as long-distance signals that regulate growth of
tubers in potato.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A-1B depict the screening of StBEL11 (FIG. 1A) and
StBEL29 (FIG. 1B) CaMV 35S transgenic overexpression lines using
RT-qPCR off RNA from in vitro plantlets. GAPDH RNA was used as an
internal control. The lines designated with arrows were used for
phenotypic analysis. Values shown in the bar graphs are relative to
wild-type andigena levels.
[0028] FIGS. 2A-2B depict the screening of GAS:StBEL11 and
GAS:StBEL29 transgenic lines. RNA was extracted from transgenic in
vitro plantlets and one-step RTPCR (FIG. 2A) or RT-qPCR (FIG. 2B)
with GSPs were performed for StBEL11 and StBEL29, respectively.
Only transgenic StBEL11 RNA was assayed in FIG. 2A. Values shown in
FIG. 2B are relative to WT andigena levels. Lines 11a, b, c, and e
(FIG. 2A) and StBEL29 lines 9 and 19 (FIG. 2B) were identified as
high expressers with significant phenotypes and were used in
subsequent experiments.
[0029] FIGS. 3A-3D show 35S:antisense RNA lines screened for
StBEL11 or StBEL29 RNA from either leaves of one-month old
soil-grown plants or stolons from 21 day short-day plants. Lines
designated 11-1 (C-72), 11-2 (C-75), 29-1 (C-9A), and 29-2 (C-1B)
were used for further analyses. RT-qPCR with gene-specific primers
was used for the quantification. Each sample was measured in
duplicate. Details of the transformation and screening protocols
are described in the examples infra.
[0030] FIGS. 4A-4B depict shoot (FIG. 4A) and tuber (FIG. 4B)
yields in 7540 (WT), 35S:StBEL11 (lines 7, 11) and 35S:StBEL29
(lines 6, 11) overexpression lines. Error bars
represent.+-.standard deviation of seven biological replicates. A
Student's t test was performed to check significance with one, two,
and three asterisks indicating p<0.05, p<0.01, and
p<0.001, respectively. Plants were grown under long-days for
4-weeks, followed by 4-weeks under short-day conditions. ns
indicates not significant.
[0031] FIGS. 5A-5C are graphs showing phenotypes of antisense lines
for StBEL11 (11-1, 11-2) and StBEL29 (29-1, 29-2). After screening,
selected independent lines were grown for 8-weeks under long-days
and then 21 days under short-day conditions. Ten independent plants
for each transgenic line, including wild-type, were used for
evaluating shoot growth (FIG. 5A) and tuber yields (FIG. 5B) at 21
days under short-days. Error bars represent.+-.SD (n=10). 7540 is a
non-transformed WT line. For analysis of the tuber marker gene
StSP6A (FIG. 5C), RNA was harvested from stolons of plants grown
under short-days for 21 days and StSP6A was quantified using
RT-qPCR with gene-specific primers in selected lines for both
types. Stolons were pooled from three plants off ten independent
plants per transgenic line forming three biological replicates per
line for RT-qPCR analysis. Three technical replicates were
performed for each biological replicate for RT-qPCR. Error bars
represent.+-.standard deviation from n=3. A Student's t test was
performed to check significance with one, two, and three asterisks
indicating p values of <0.05, <0.01, and <0.001,
respectively. ns indicates not significant.
[0032] FIGS. 6A-6C show results of tubers from 35S:antisense RNA
lines for StBEL11 and StBEL29 (FIG. 6A). Plants were grown in a
growth chamber for eight weeks under long days and then 21 days
under short-day conditions before harvest. 7540 is a
non-transformed wild-type line. For each line, tubers are pooled
from three plants. White bar=1.0 cm. The mean number of tubers per
plant are shown for antisense lines of StBEL11 (FIG. 6B) and
StBEL20 (FIG. 6C). The arrows in FIGS. 6B-6C indicate the lines
shown in the photo (FIG. 6A).
[0033] FIGS. 7A-7I show Promoter:GUS constructs of upstream
sequence of StBEL11 and StBEL29. The scale bar in FIG. 7A is
equivalent to 200 bp. GUS expression driven by promStBEL11 (FIGS.
7B, 7D, and 7E) and promStBEL29 (FIGS. 7F-7H) in 2 week old in
vitro transgenic plants. GUS expression was observed in the
midvein, petioles, and stems. Both promoter fusion lines were also
grown in soil for 8-weeks in growth chambers under long-days and
were then subjected to short-day conditions for 15 more days. GUS
expression is shown in stolon from promStBEL11 (FIG. 7C, arrow) and
swollen stolon from prom-StBEL29 (FIG. 7I, arrows). Scale bar
equivalent to 0.5 cm in FIGS. 7B and 7H, and 500 .mu.m for FIGS.
7D-7G.
[0034] FIGS. 8A-8D show GUS activity in 8-week soil-grown
proStBEL11:GUS (FIGS. 8A and 8C) and proStBEL29:GUS transgenic
lines (FIGS. 8B and 8D) cultured under long-day conditions for 6
weeks and then under short-day conditions for two-weeks. Transverse
sections of petioles (FIGS. 8A and 8B) and stems (FIGS. 8C and 8D)
of proBEL11:GUS (FIGS. 8A and 8C) and proBEL29:GUS (FIGS. 8B and
8D) transgenic lines. The arrows in FIGS. 8A and 8B designate
external phloem cells. The arrows in FIG. 8C designate xylem cells
and the epidermis. The arrow in FIG. 8D designates vascular
bundles. X indicates xylem cells.
[0035] FIGS. 9A-9D show relative accumulation of total (FIGS. 9A
and 9B) and polysomal (FIGS. 9C and 9D) RNA of StBEL11 (FIGS. 9A
and 9C) and StBEL29 (FIGS. 9B and 9D) in WT potato (Solanum
tuberosum ssp. andigena) plants under short-day and long-day
photoperiods. RT-qPCR was performed on RNA from leaves, petioles,
stem, roots, and stolons from 8-week soil-grown plants incubated
under short-day (white bar) and long-day (black bar) for 15 days.
Data represented is normalized with GAPDH. Ct value for long-day
leaves set at 1.0. The values shown are the average of three
biological replicates.+-.standard deviation. A Student's t test was
performed to check significance with one and two asterisks
indicating p<0.05 and p<0.01, respectively.
[0036] FIGS. 10A-10B show the effect of photoperiod on promoter
activity of StBEL11 (FIG. 10A) and -29 (FIG. 10B). Tissue specific
GUS activity in proStBEL11:GUS and proStBEL29:GUS lines was
measured under both long-day (16 h light, 8 h dark) and short-day
(8 h light, 16 h dark) conditions. Both promoter fusion lines were
grown in soil for 8 weeks in growth chambers under long days and
were then subjected to either long-day (black bars) or short-day
(open bars) conditions for 15 more days. Quantification was
performed using a fluorometric assay with
4-methylumbelliferyl-.beta.-D-glucuronide as substrate. Data
represent the mean.+-.standard deviation of GUS activity measured
in three biological replicates. GUS activity is expressed as nmol
4-methylumbelliferone .mu.g protein.sup.-1 hr.sup.-1. A Student's t
test was performed to check significance with one and two asterisks
indicating p<0.05 and p<0.01, respectively. Quantitative GUS
activity in respective tissues (long-day) were used as a reference
in statistical analyses of both panels (FIGS. 10A and 10B).
[0037] FIGS. 11A-11D show movement of StBEL11 (FIG. 11A) and
StBEL29 (FIG. 11B) mRNA across heterografts of soil-grown plants.
For heterografts, micrografts were performed with four replicates
of GAS:StBEL11 (line 11b), GAS:StBEL29 (line 29-9) and GAS: GUS
scions on wild-type andigena stocks. After 2 weeks in culture,
grafts were moved to soil and grown under long-days for three weeks
and then under short-days for 2 weeks before harvest of leaves,
secondary roots, and stolons. Following RNA extraction,
gene-specific primers (GSP) were used with a non-plant DNA tag
specific for the transgenic RNA (designated NT-2 in the examples
herein) to perform one-step RTPCR on 250 ng of total RNA from
wild-type secondary roots and stolons of all four heterografts
(FIGS. 11A and 11B). RNA from scion leaf samples was used as a
positive control (scion samples). All PCR products detected in
scion (positive control) and stock (test for movement) RNA samples
represent transgenic RNA. GSPs for transgenic StBEL11 (FIG. 11A),
transgenic StBEL29 (FIG. 11B), and transgenic GUS (FIG. 11C) were
used. Heterografts are designated R1-4 (root stock RNA) and St1-4
(stolon stock RNA). RNA from secondary roots and stolons of
wild-type/wildtype (andigena) autografts was used in the RT-PCR
with transgenic StBEL11 and -29 gene-specific primers as a negative
control (FIG. 11D). Similar negative results were obtained with RNA
from wild-type leaves.
[0038] FIG. 12 is a graph showing the photoperiod effect on the
movement of transgenic StBEL29 and StBEL11 mRNA into stolons.
Movement of transgenic mRNA from leaf to stolon was quantified in
transgenic lines expressing full length StBEL11 and -29. Expression
was driven by the galactinol synthase (GAS) promoter of melon
(Cucumis melo). This promoter is selectively active in the minor
veins of leaf mesophyll (Ayre et al., "Functional and Phylogenetic
Analyses of A Conserved Regulatory Program in the Phloem of Minor
Veins," Plant Physiol. 133:1229-39 (2003) and Banerjee et al.,
"Untranslated Regions of a Mobile Transcript Mediate RNA
Metabolism," Plant Physiol. 151:1831-43 (2009), which are hereby
incorporated by reference in their entirety). Relative levels of
transgenic RNA were quantified from RNA extracted from 0.5 cm
samples from the tip of the stolon. GAS:StBEL11 and -29 plants were
grown under long-day conditions for 4 weeks and then transferred to
either short-days (SD) for two-weeks or maintained under long-days
(LD). RT-qPCR with gene-specific primers was used to measure the
relative amounts of transgenic RNA in stolons. Each of the stolon
values has been calculated relative to a value of 1.0 for the
amount of transgenic RNA measured in either LD or SD leaves from
the same plant. Stolons from two plants off four independent plants
for each construct were pooled forming two biological replicates.
Each biological replicate was measured with two technical
replicates and normalized against StActin8 mRNA. The fold change in
RNA levels was calculated as the 2.sup.-.DELTA..DELTA.ct value.
Standard errors of the means of two biological replicates are shown
with one, two and three asterisks indicating significant
differences (p<0.05, p<0.01, p<0.001, respectively) using
a Student's t test.
[0039] FIGS. 13A-13B show shoot (FIG. 13A) and tuber (FIG. 13B)
yields in 7540 (WT) and GAS:StBEL11 and -29 transgenic lines grown
under short-day conditions. Data represent the mean of four
biological replicates (n=4). One or three asterisks indicate
significance (p<0.05 and p<0.001, respectively) using a
Student's t test. Plants were grown under long-days for 4 weeks,
followed by 3 weeks under short-day conditions.
[0040] FIG. 14 shows tuber yields of StBEL29 overexpression lines 9
and 19. Both transgenic lines expressed StBEL29 using the
leaf-specific GAS promoter. Six replicates per line were assessed
for tuber production after 21d short-day conditions. The data
represent the mean.+-.SD. Line 7540 is untransformed Solanum
tuberosum ssp andigena.
[0041] FIGS. 15A-15B show relative levels of StSP6A and StPIN1 RNA
in tuberizing stolons of WT 7540 andigena (open bars), or
GAS:StBEL11 and -29 transgenic lines (black bars) grown under
short-day conditions. Plants were grown under long-days for 4
weeks, followed by 2 weeks under short-day conditions. Stolons from
two plants off four independent plants for each construct were
pooled forming two biological replicates (n=2). Each biological
replicate was quantified using two technical replicates.
[0042] FIG. 16 shows relative levels of StBEL5 RNA in tuberizing
stolons of 7540 andigena (WT), GAS:StBEL11 (G:B11), and -29 (G:B29)
transgenic lines grown under short-day conditions. Data represent
the mean.+-.standard deviation of two biological reps and two
technical reps. Plants were grown under long days for four weeks
followed by two weeks under short-day conditions.
[0043] FIGS. 17A-17D show RNA movement assays for StBEL11 (FIGS.
17A and 17B) and StBEL29 (FIGS. 17C and 17 D) into roots (FIGS. 17A
and 17C) and stolons (FIGS. 17B and 17D) using the PVX-based system
with over-expression lines of the RNA-binding proteins, StPTB1 and
StPTB6, 8 days post-inoculation of leaves. RNA was extracted and
RT-qPCR with gene-specific primers was used to calculate the
relative amounts of StBEL11 or StBEL29 RNA. Each sample was
measured and normalized against StACtin8 RNA. RNA values were
calculated as the 2.sup..DELTA..DELTA.Ct value relative to the mean
values obtained from WT samples. Standard deviations of the means
of two biological replicates with two technical replicates are
shown with one, two, and three asterisks indicating significant
differences (p<0.05, p<0.01, p<0.001, respectively) using
a Student's t-test. This system was used previously to monitor
StBEL5 RNA movement (Cho et al., "Polypyrimidine Tract-Binding
Proteins of Potato Mediate Tuberization Through an Interaction With
StBEL5 RNA," J. Expt. Bot. 66:6835-47 (2015), which is hereby
incorporated by reference in its entirety). Movement is facilitated
in the StPTB overexpression lines.
[0044] FIG. 18 shows that StBEL5 induces StBEL11 and StBEL29 in
stolons from GAS:BEL5 transgenic lines grown under short-day
conditions for 12 days. RT-qPCR in replicate was performed on the
RNA extracted from the 12 day stolons. StBEL5 RNA levels are
included as a control. The GAS promoter is specific to the minor
veins of the leaf mesophyll (Ayre et al., "Functional and
Phylogenetic Analyses of A Conserved Regulatory Program in the
Phloem of Minor Veins," Plant Physiol. 133:1229-39 (2003), which is
hereby incorporated by reference in its entirety) and enhanced
levels of StBEL5 RNA in stolons are the result of movement of
StBEL5 RNA from leaves to stolons (Banerjee et al., "Dynamics of a
Mobile RNA of Potato Involved in a Long-Distance Signaling
Pathway," Plant Cell 18:3443-57 (2006); Banerjee et al.,
"Untranslated Regions of a Mobile Transcript Mediate RNA
Metabolism," Plant Physiol. 151:1831-43 (2009); and Lin et al.,
"The Impact of the Long-Distance Transport of a BEL1-Like mRNA on
Development," Plant Physiol. 161:760-72 (2013), which are hereby
incorporated by reference in their entirety). In this model, mobile
StBEL11 and StBEL29 contain the tandem TGAC motif specific for the
BEL/KNOX complex in their upstream sequence. Data represent the
mean.+-.standard deviation of two biological reps and two technical
reps. One or three asterisks indicate a significant difference at a
0.05 or 0.001 level, respectively, using a Student's t test.
[0045] FIG. 19 shows an amino acid sequence alignment of StBEL5
(SEQ ID NO:51), StBEL11 (SEQ ID NO:52), and StBEL29 (SEQ ID NO:53).
Black- and gray-boxed letters represent identical or similar
residues, respectively. The conserved BELL domain and the
N-terminal Sky and C-terminal VSLTLGL boxes have been underlined.
The homeodomain is marked by a dashed line. The amino acids for
conserved domains are aligned in relation to the BEL5 protein.
Ovals highlight areas of eight residues or more of unique
sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Generally, the present invention relates to nucleic acid
molecules encoding BEL transcription factors from potato (Solanum
tuberosum L.), BEL transcription factor is a general term used
herein to mean a member of the BEL-1-like family of transcription
factors, which includes a BELL domain (Bellaoui et al., "The
Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact
Through a Domain Conserved Between Plants and Animals," Plant Cell
13(10:2455-70 (2001), which is hereby incorporated by reference in
its entirety) and which regulates growth, in particular, floral
development.
[0047] A description of the BEL mobile mRNA and their regulation of
tuber development in potato including the mechanisms mediating
their mobility are further described in Hannapel and Banerjee,
"Multiple Mobile mRNA Signals Regulate Tuber Development in
Potato," Plants 2017: 6, S (2017), which is hereby incorporated by
reference in its entirety.
[0048] One aspect of the present invention is directed to a nucleic
acid construct comprising a first nucleic acid molecule comprising
a nucleotide sequence configured to silence or reduce expression of
StBEL11 and variants thereof; a 5' DNA promoter sequence; and a 3'
terminator sequence, where the first nucleic acid molecule, the
promoter sequence, and the terminator sequence are operatively
coupled to permit transcription of the first nucleic acid
molecule.
[0049] The mRNA of StBEL11 has a nucleotide sequence of SEQ ID
NO:1, as follows, where the 5' UTR and 3' UTR are shown in italics,
preceding and following the CDS, which is shown in regular
font:
TABLE-US-00001 tttaagaaaa tctctcactt tctctttctc ccaattataa
taagaaaact ttctttcctc 60 cttgttttta tttttaaaaa aatatttcag
tttagtttat ggttgaagat atttgatata 120 gccttcatat atgtcactca
tgttccatca tcagccaagt gttagaagtc actttcttta 180 acaagatttt
cttgaaaaat atttaaaaaa ttgaactcca aaaaaaagaa aaaaaggagt 240
gtagttttct tgattggttg tgaaatttat ggctatgtac tatcaaggag gctcagaaat
300 ccaagctgat ggtctgcaga cactttattt gatgaaccct aactatatag
gctacactga 360 cacacatcat catcatcatc aacaccaaca acaatcagcc
aacatgtttt tcttgaattc 420 tgtggcggcg gggaattttc cccacgtgtc
cctccctttg caagcacatg cgcaggggca 480 cttggttgga gtgcccctgc
cagctggttt tcaagatcct aaccgccctt ccattcagga 540 aattccgacc
tctcatcatg gccttttatc gcgtttgtgg acttctggtg accaaaatac 600
ccctagaggt ggtggaggag gaggagaagg aaatggaagt caatcacata taccgtcttc
660 cacggtggtt tctcccaact caggtagtgg gggaggcacc accacggact
ttgcttccca 720 attagggttc caaagaccgg ggttggtgtc accaacacag
gcgcaccatc aaggtctttc 780 tctaagcctt tctccacaac aacaaatgaa
tttcaggtct agtcttccac tagaccaccg 840 cgatatttca acaacaaatc
atcaagttgg aatactatca tcatcaccat taccatcacc 900 aggaacaaat
accaatcata ctcgaggatt aggggcatca tcgtcttttt cgatttctaa 960
tgggatgata ttgggttcta agtacctaaa agttgcacaa gatcttcttg atgaagttgt
1020 taatgttgga aaaaacatca aattatcaga ggttggtgca aaggagaaac
acaaattgga 1080 caatgaatta atatctttgg ctagtgatga tgttgaaagt
agcagccaaa aaaatagtgg 1140 tgttgaactt actacagctc aaagacaaga
acttcaaatg aagaaagcaa agcttgttag 1200 catgcttgat gaggtggatc
aaaggtatag acaataccat caccaaatgc aaatgattgc 1260 aacatcattt
gagcaaacaa caggaattgg atcatcaaaa tcatacacac aacttgcttt 1320
gcacacaatt tcaaagcaat ttagatgttt aaaagatgca atttctgggc aaataaagga
1380 cactagcaaa actttagggg aagaagagaa cattggaggc aaaattgaag
gatcaaagtt 1440 gaaatttgtg gatcatcatt tacgccaaca acgtgcacta
caacaattag ggatgatgca 1500 aaccaatgca tggaggccac aaagaggttt
gcccgaaaga gcggtttcgg ttctccgtgc 1560 ttggcttttc gagcattttc
ttcatccgta tcccaaagat tcagataaaa tcatgcttgc 1620 taagcaaaca
gggctaacaa ggagccaggt atcaaattgg tttataaatg ctagagttag 1680
actatggaag ccaatggtag aagaaatgta catggaagaa gtgaagaaaa acaatcaaga
1740 acaaaatatt gagcctaata acaatgaaat tgttggttca aaatcaagtg
ttccacaaga 1800 gaaattacca attagtagca atattattca taatgcttct
ccaaatgata tttctacttc 1860 caccatttca acatctccga cgggcggcgg
cggttcgatt ccggctcaga cggttgcagg 1920 tttctccttc attaggtcat
taaacatgga gaacattgat gatcaaagga acaacaaaaa 1980 ggcaagaaat
gagatgcaaa attgttcaac tagtactatt ctctcaatgg aaagagaaat 2040
catgaataaa gttgtgcaag atgagacaat caaaagtgaa aagttcaaca acacacaaac
2100 aagagaatgt tattctctaa tgactccaaa ttacacaatg gatgatcaat
ttggaacaag 2160 gttcaacaat caaaatcatg aacaattggc aacaacaaca
acaacttttc atcaaggaaa 2220 tggtcatgtt tctcttactc tagggcttcc
accaaattct gaaaaccaac acaattacat 2280 tggattggaa aatcattaca
atcaacctac acatcatcca aatattagct atgaaaacat 2340 tgattttcag
agtggaaagc gatacgccac tcaactatta caagattttg tttcttgatg 2400
atatatataa tttgcaggta aatcagcttg aaattacatc atgaaaggcc ttgaataaaa
2460 gaaggggagt tgagatctag tgatcatata aatatgtata ggtagaaagt
ttagttagta 2520 tatataggtt atacttctag tttcttaaat ggagatacaa
tttttgttgt tatttttgta 2580 ttgagataac tagctagctt ggattattta
aagttgttgc atgcaaccaa agaagaagaa 2640 aaaataatct atatatgcaa
actatagtat gttgtaaatt ttgtgcgtct ttttgtttca 2700 atttgcatat
atgtaaac 2718
[0050] The mRNA of StBEL11 described above is derived from StBEL11,
having a nucleotide sequence of SEQ ID NO:2, as follows, where the
upstream sequence is shown in italics, introns are shown in regular
font, and exons are shown in bold:
TABLE-US-00002 ttttttttat gtatatatac atttgatgaa gataatgttc
tcttaagtga aaatcttgct 60 tttatcatta gttagtactt acaattcttt
ctgtcttatt ttatatgata tttttttaaa 120 tttagtttac cccgaaaata
aatgatatgt ttttatatat ttaactaatt caatttaact 180 aattcaattt
taaacttctt tgaatctcaa tcgaattgcc tcatttttga gaaggagttc 240
gatttcaaac ccagattcga tccactccaa gaaaaagaga aaagaaaaac aaatcaacta
300 cgaaccccca ccccacccca ccccaccccc caccatcgga aaaagggtca
taagtagaaa 360 taaagaaaaa ttgagggact tctagcaact aatgtaatca
attatgtatt atatatggac 420 ccaacaaatt ggtggaaaaa gacgtttcct
catttttcat atatctatgg cctacttcct 480 ttaagttaat gttttttttc
ttcatctaat tttaagtcga gtatttattt tgagactcgg 540 attaatttaa
attgatgttt tcaggaaaat ttatcaaaag tgaaaatcta acttattgag 600
aattttctta tttgtatgat ttaaatttgt aacctctaaa taaagatgaa aaatcttaat
660 catttcatca ttactcgtaa ttattttctt cttgttagtg ttcactatac
tctctctttc 720 tctctaaaga tatttttgaa aaaaaatatc taaattatgc
cagcatcaaa tcattttata 780 atagtgaaat taagattggg tctatttatt
ttttccatca cacgtatgta gaacccccca 840 cccccaccct cgccgccacc
ccaccccctt actatcgagt ttaactaata tttattagta 900 taaaaattat
atttatctgt tataacaagt aaaatgtctt atttttaaaa ggataaaggt 960
atgagaaata tcccaacttt gatcggattt actgttgcga tactaaactt tcatgaggat
1020 ctattacctc cttcgactat ttaataccgt atttttatcc ccctgaacta
tttaatattg 1080 tattttaaag gtatatatga ttatatgtgc caacgtggac
acattactat ttataatttt 1140 gcattatttt ttatgtccac gtggacaaat
atatatgttt aaaatacggt attaaatagt 1200 ctagggagct aataggtcct
catgaaagtt tagtatcgca acaacaaatt cgatcaaagt 1260 tgagatattt
ttcaggccct tatccctatt tttaaaattg aaagtttaca tttttatgaa 1320
gggttaaaac atgtaacatc atttaggtaa cttgatatag tataaaaaat tatttacatt
1380 atatataaat taaattcatg attactaaaa gaattcaatc atcaggtcat
ctttatctat 1440 gaaatgtttt atttgtaaaa ttacaaacct cacatttaaa
aaagtttatc tataaatata 1500 tttttaaata accttcctga taatgtaaaa
atatttatac tgacgattct tactgatttt 1560 ttttttactg tgtttttgag
gggtggggtg ggggtgaggg taagggggat atgttgggag 1620 acttacacta
aataaacatg tcttctttat tcatattccc ctttatgtgt tgtggagttt 1680
taagaaaatc tctcactttc tctttctccc aattataata agaaaacttt ctttcttcct
1740 tgtttttatt tttaaaaaaa tatttcagtt tagtacatgg ttgaagatat
ttgatatagc 1800 cttcatatat gtcactcatg tgagtacaac ttttctccat
atatatcaaa atcaagattt 1860 tcatagttga gtgattaatt aattgtatat
aactcatcat atattatttg aattttcttt 1920 gttaaaaatg ttttctatct
ttagggtatt gcatggattt attataattt ttttctatct 1980 tactttctaa
tttcaggttc catcatcagc caagtgttag aagtcacttt ctttaacaag 2040
attttcttaa aaaatattta aaaacttgaa ctccaaaaaa aagaagaaaa ggagtgtaat
2100 tttcttgatt ggttgtgaaa tttatggcta tgtactatca aggaggctca
gaaatccaag 2160 ctgatggtct gcagacactt tatttgatga accctaatta
tataggctat actgacacac 2220 atcatcatca tcaacaacac caacaacaat
cagccaacat gtttttcttg aattctgtgg 2280 cggcggggaa ttttccccac
gtgtccctcc ctttgcaagc acatgcgcag gggcacttgg 2340 ttggagtgcc
cctgccagct ggttttcaag atcctaaccg cccttccatt ccggaaattc 2400
cgacctctca tcatggcctt ttatcacgtt tgtggacttc tggtgaccaa aataccccta
2460 gaggtggtgg aggaggagga gaaggaaatg gaagtcaatc acatataccg
tcttccacgg 2520 tggtttctcc caactcaggt agtgggggag gcaccaccac
ggactttgct tcccaattag 2580 ggttccaaag accggggttg gtgtcaccaa
cacaggcgca ccatcaaggt ctttctctaa 2640 gcctttctcc acaacaacaa
atgaatttca ggtctagtct tccactagac caccgcgata 2700 tttcaacaac
aaatcatcaa gttggaatac tatcaccatc accattacca tcaccaggaa 2760
caaataccaa tcatactcga ggattagggg catcatcgtc tttttcgatt tctaatggga
2820 tgataatggg ttctaagtac ctaaaagttg cacaagatct tcttgatgaa
gttgttaatg 2880 ttggaaaaaa catcaaatta tcagagggtg gtgcaaagga
gaaacacaaa ttggacaatg 2940 aattaatctc tttggctagt gatgatgttg
aaagtagcag ccaaaaaaat attgttgttg 3000 aacttactac agctcaaaga
caagaacttc aaatgaagaa agccaagctt gttagcatgc 3060 ttgatgaggt
atatatactt ctaattattc atatattaat taattaatca tatatatata 3120
ttaatcaaat tattcatata ttaattaatt aatcaatacc aagtttcttg atttggagtt
3180 tgatcattta ggcaaatttc actactatat ataaaacaca aattctaacg
gatgtttggt 3240 cagtaagaaa ttctcaattt tcaatcaaac atctattaga
atttcacgtt ttttagtagt 3300 gaaattaatt aaataatcta aaaattgttc
aatcgaattt acaacaaaac atccattgaa 3360 attttacttt cttttcaata
gcgaaaccaa ttaaatagga ttgactaccc aattaatagt 3420 tattatctta
tcttcttctt gatttcaatc ttttttcaat aaaaagagta attttaatta 3480
tgagataata aactactgat tagttatgac aatctgaaaa atcaactcct attaaatgat
3540 ccaaaaagtg tacaaatttg tatatcttaa tgttaatttc attgttttat
ttatttattt 3600 agttgctttt ttttccttct tgggaagggg gaggggtcaa
gttgctattg attcatatac 3660 tagcaataat tattgattta tttcaaaggt
acaattttgt tcatcatgaa actataagct 3720 agacaatata tgtggttcta
agcttttttc tattgggggt ccaactaaag ttaaagataa 3780 tacacctaat
atgtcttgtt gagttgacaa aaaatcaaag gcacgtggtc ttattcaatc 3840
actttattag aacctttcaa atttggaaat attcttacct ttcttttgag ataatcacat
3900 aaaaataaac ctttggaata atttatattt ttggtattcc tattgatatt
tgatatctgt 3960 tttgaagctt aactaatata aatatgcgtc gaaaagttat
ctcactttag gagattaaaa 4020 tgcttcttag taaaagtgac ttcatattca
ggactcgaat aatattactt gatgatcatt 4080 tggacacaat tgatcaattg
ggaaataatg aaatattatt ttgcaaatca agttttattt 4140 tataatttca
ttagttattt cgacttgaac ttgaaataaa gaatctgaag tttgaaaaat 4200
tgatttaaag agtatttttt tccactctaa gaacttcaaa caatttcaac ttcaacttca
4260 tataatcata ttttttttca acttcaatca gatatcgtcg tgatatgatc
aatatcaatc 4320 tacttatttt tattttattg tttgtatttt tttttgaatt
ttttataggt ggatcaaagg 4380 tatagacaat accatcacca aatgcaaatg
attgcaacat catttgagca aacaacagga 4440 attggatcat caaaatcata
cacacaactt gctttgcaca caatttcaaa gcaatttaga 4500 tgtttaaaag
atgcaatttt tgggcaaata aaggacacaa gtaaaacttt aggggaagaa 4560
gagaacattg gaggcaaaat tgaaggatca aagttgaaat ttgtggatca tcatttacgc
4620 caacaacgtg cactacaaca attagggatg atgcaaacca atgcatggag
gccacaaaga 4680 ggtttgcccg aaagagcggt ttcggttctc cgcgcttggc
ttttcgagca ttttcttcat 4740 ccgtaagtat ttgttgaaga cataattaag
taaattaata tgcatgtctt ttaatagttt 4800 aagattttaa acaaagcaat
cacaacatcc tacatgtttc accgcttgtt ctccttatta 4860 ggaaaaataa
ccaattgttc tagagtatat gagaaagaat cagactcgca atctagcatt 4920
tgaagtggca aatacaagac taattaagta aatacaattt ttttttttaa aataacagtt
4980 taaacttttg aatgagatag atttaattaa caccttatat tacctataag
aaatgaactt 5040 caatctctat ttttttttta aaaacaattt tatacaccat
gtagaaacct ttataaagaa 5100 attaaattaa atcactcata ccatttcttt
taaatttcaa taaataaatt atatatttct 5160 tgtcttgcag gtatcccaaa
gattcagata aaatcatgct tgctaagcaa acagggctaa 5220 caaggagcca
ggttcttgaa aaattcatca tctcaattta tatgacgcat tttttaacat 5280
atctaaaaaa gacgttttat ttctaattta gaaacaataa aattttaaaa ttctcaacaa
5340 tcatagtacc tctctatata actgtaacaa catcttatta taacaaccaa
ttttttgaat 5400 atgccgtaaa aaagacatta tattttcaat ttaggaacaa
tataacttta aaattctcaa 5460 caatcatagt acctctctat ataactgtaa
caacatctta ttataacaac caattttttg 5520 aatatgccgt aaaaaagaca
ttatattttc aatttaggaa caatataact ttaaaattct 5580 caacaatcat
agtacctctc tatataacag taacaacatc ttgttataac aaactaagtt 5640
cttttttaaa ccaactttca ctacaacaaa gataactttt agcggcaata tacatattaa
5700 taaagaatac taaagctttt accggcatta gttaatttca ttggatccat
tatcgctata 5760 gactgtagat acatttacaa aaagtattaa ttaccactaa
aaacacatat gtagtggcaa 5820 ttttgctatt gctattaatt aattaatgtt
ataaatataa tttttaatgt agtgtttcat 5880 gttatgttaa agtcatgtag
tatatgttct ctacgaataa tatttcacta tatcagacaa 5940 aaaatatcta
gaataaacaa tgatgttata gaaagatttg acagcaagtc acacaaatat 6000
gtacttaaga gtacttattt tagactacaa gttttaaaag tcgatcgtct gttctttctt
6060 aaaatacttt tgaaaatgca ggtatcaaat tggtttataa atgctagagt
tagactatgg 6120 aagccaatgg tagaagaaat gtacatggaa gaagtgaaga
aaaacaatca agaacaaaat 6180 attgagccta ataacaatga aattgttggt
tcaaaatcaa gtgttccaca agagaaatta 6240 ccaattagta gcaatattat
tcataatgct tctccaaatg atatttctac ttccaccatt 6300 tcaacatctc
cgacgggtgg cggcggttcg attccggctc agacggttgc aggttagttg 6360
gaatataaag aaagtcattt taaaagttgt cgttgtttga cctataatag gttttgagtc
6420 gtggaaggca tcactaattt gcataaaggt aggttgtccc cttggggtat
ggtcttttca 6480 tggagtaacg gtagagttgt ttccactgac ctatataagt
tacaggttcg agttgtggaa 6540 ttggttgcgt tgttgatgct catgtcgggg
tagactgtct acaacacaca ccttgagata 6600 caacctttta ttgtacccta
catgaatgtg aaatacttca cgcaccaaac tgcctaataa 6660 ctcttagaaa
agaacacact tgactcacac atctatatat ctacgtagta cctcaattga 6720
taaataatct aggatgatta gatggttaca catatcaaac atataatagg ttcaagtcgt
6780 agaaggcgtc actaatttgc atcaaggtag gttgtcccct tggggtatga
tcctttcatg 6840 gaccatgtag acactcttga agttgagtct tgaagtaaca
gtaaagtcat ctccacgtga 6900 cctatatata taagtcacaa cttcgagttg
tggagttggc taggctctca tcaggataga 6960 ctgtcgacat cacacttctt
gaaatgcaac cttttttcga accttatgtg aatgtgagac 7020 tacactcttg
actaacatct atataactac tatacctcaa ttaataaaca atctaggata 7080
attagatggt ctcacactct aaacacctag gttagatcaa aagacaataa aactagctag
7140 agtacatttt tatttattgt aacaagtgtt acttatcaaa gtgtgactct
atattgttta 7200 actaattaac atgtttaatt tgtctaaaca ggtttctcct
tcattaggtc attaaacatg 7260 gagaacattg atgatcaaag gaacaacaaa
aaggcaagaa atgagatgca aaattgttca 7320 actagtacta ttctctcaat
ggaaagagaa atcatgaata aagttgtcca agatgagaca 7380 atcaaaagtg
aaaagttcaa caacacacaa acaagagaat gctattctct aatgactcca 7440
aattacacaa tggatgatca atttggaaca aggttcaaca atcaaaatca tgaacaattg
7500
gcaacaactt ttcatcaagg aaatggtcat gtttctctta ctctagggct tccaccaaat
7560 tctgaaaacc aacacaatta cattggattg gaaaatcatt acaatcaacc
tacacatcat 7620 ccaaatatta gctatgaaaa cattgatttt cagagtggaa
agcgatacgc cactcaacta 7680 ttacaagatt ttgtttcttg atgatatata
taatttccag gtaaatcaac ttgaaattac 7740 atcatgaaag gccttgaata
aaagaagggg agttgagatc tagtgatcat atatatatgt 7800 ataggtagaa
agtttagtta gtatatatag gttatacttc tagtttctta aatggagata 7860
caatttttgt tgttgttttt gtattgagat aactagctag cttgggttat ttaaagttgt
7920 tgcatgcaac caaagaagaa gaaaaaataa tctatatatg caaactatag
tatgttgtaa 7980 attttgtgct tcttttaatt agtttcaatt tgcatatatg taaac
8025
[0051] According to one embodiment, the nucleic acid construct of
the present invention comprises DNA heterologous to the first
nucleic acid molecule. In one particular embodiment, the DNA
heterologous to the first nucleic acid molecule is the 5' DNA
promoter sequence.
[0052] In one embodiment, the first nucleic acid molecule
comprising a nucleotide sequence configured to silence or reduce
expression of StBEL11 has a nucleotide sequence of SEQ ID NO:3, as
follows:
TABLE-US-00003 gaaatttatg gctatgtact atcaaggagg ctcagaaatc
caagctgatg gtctgcagac 60 actttatttg atgaacccta actatatagg
ctacactgac acacatcatc atcatcatca 120 acaccaacaa caatcagcca
acatgttttt cttgaattct gtggcggcgg ggaattttcc 180 ccacgtgtcc
ctccctttgc aagcacatgc gcaggggcac ttggttggag tgcccctgcc 240
agctggtttt caagatccta accgcccttc cattcaggaa attccgacct ctcatcatgg
300 ccttttatcg cgtttgtgga cttctggtga ccaaaatacc cctagaggtg
gtggaggagg 360 aggagaagga aatggaagtc aatcacatat accgtcttcc a
401
[0053] Other nucleic acid molecules may also be used as the first
nucleic acid molecule comprising a nucleotide sequence configured
to silence or reduce expression of StBEL11. For example, in certain
embodiments, the first nucleic acid molecule has a nucleotide
sequence that is at least about 95%, 96%, 97%, 98%, or 99%
identical to the nucleotide sequence of SEQ ID NO:3.
[0054] By way of other examples, the first nucleic acid molecule
has a nucleotide sequence that is at least about is at least 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% identical to the
nucleotide sequence of SEQ ID NO:3.
[0055] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous bp of SEQ
ID NO:3.
[0056] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous bp of SEQ
ID NO:2 which are less than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10%, 5%, or 1% identical to any portion of SEQ
ID NO:8, where the portion of SEQ ID NO:8 has the same length as
the first nucleic acid molecule.
[0057] According to this embodiment, one can silence or reduce
expression of StBEL11 without silencing or reducing the expression
of StBEL5.
[0058] Methods for identifying nucleic acid molecules capable of
silencing or reducing expression of genes and/or related RNA
molecules are well known in the art and are discussed in more
detail infra.
[0059] Nucleic acid molecules capable of silencing or reducing
expression of StBEL11, using any of the methods described infra are
contemplated.
[0060] In a further embodiment, the nucleic acid construct
according to this aspect of the present invention further comprises
a second nucleic acid molecule comprising a nucleotide sequence
configured to silence or reduce expression of StBEL29 and variants
thereof.
[0061] The mRNA of StBEL29 has a nucleotide sequence of SEQ ID
NO:4, as follows, where the 5' UTR and 3' UTR are shown in italics,
and the CDS is shown in regular font:
TABLE-US-00004 ttctttcttt ctttctcctc tctctctctc taaaaagttg
agtactttta ttagctctca 60 tcacttcaca cagaagaaga tggtattttt
atttctttct gctgatggct gcatcaaatg 120 atttgaaaag ctgagtcaaa
tcagaagaag aaaaagaaag ttataataat aataatgata 180 atatcaaaaa
tattattttc agattagttg gtgttatttg tttattgtgg agaaaaaata 240
aattaaaaag gaagaaaaaa tggcatctta ttttcatgga aattcagaaa tacatgaagg
300 aaatgatgga ttacaaactc taatactaat gaatcctgga tatgttggat
tttctgaaac 360 acaacatcac cacgcgccac cgccgccgcc gccaggtggc
agcagcagca acatagtttt 420 cttcaactcc aatcctattg gaaattcaat
gaacttatct cacgcgccac cacctcctcc 480 accgcctcaa caacaattca
tcggtatacc cctcgccacc gccgccttca ccgccccatc 540 ccaagactcc
ggtaacaaca acaacaacga gtcaatctcc gcccttcacg gcttcctagc 600
tcgatcgtct cagtacgggt tttacaaccc ggcaaacgac ctcacggcgg cgcgtgacgt
660 cacacgcgct catcatcatc atcagcagcc aagggctttc acttacctgt
cctcgtccca 720 gcagccgggg tttgggaact tcacggcggc gcgtgagctt
gtttcttcgc cttcgggttc 780 ggcttcagct tcagggatac aacaacaaca
acagcaacaa cagagtatta gtagtgtgcc 840 tttgagttct aagtacatga
aggctgcaca agagctactt gatgaagttg taaatgttgg 900 aaaatcaatg
aaaagtacta atagtactga tgttgttgtt aataatgatg tcaagaaatc 960
gaagaatatg ggcgatatgg acggacagtt agacggagtt ggagcagaca aagacggagc
1020 tccaacaact gagctaagta caggggagag acaagaaatt caaatgaaga
aagcaaaact 1080 tgttaacatg cttgacgagg tggagcagag gtatagacat
tatcatcacc aaatgcagtc 1140 agtgatacat tggttagagc aagctgctgg
cattggatca gcaaaaacat atacagcatt 1200 ggctttgcag acgatttcga
agcaatttag gtgtcttaag gacgcgataa ttggtcaaat 1260 acgatcagca
agccagacgt taggcgaaga agatagtttg ggagggaaga ttgaaggttc 1320
aaggcttaaa tttgttgata atcagctaag acagcaaagg gctttgcaac aattgggaat
1380 gatccagcat aatgcttgga gacctcagag aggattgccc gaacgagctg
tttctgttct 1440 tcgcgcttgg ctttttgaac atttcctcca tccttatccc
aaggattcag acaaaatgat 1500 gctagcaaaa caaacaggac taactaggag
tcaggtgtcg aattggttca tcaatgctcg 1560 agttcgtctt tggaagccaa
tggtggaaga gatgtacttg gaagagataa aagaacacga 1620 acagaatggg
ttgggtcaag aaaagacgag caaattaggt gaacagaacg aagattcaac 1680
aacatcaaga tccattgcta cacaagacaa aagccctggt tcagatagcc aaaacaagag
1740 ttttgtctca aaacaggaca atcatttgcc tcaacacaac cctgcttcac
caatgcccga 1800 tgtccaacgc cacttccata cccctatcgg tatgaccatc
cgtaatcagt ctgctggttt 1860 caacctcatt ggatcaccag agatcgaaag
catcaacatt actcaaggga gtccaaagaa 1920 accgaggaac aacgagatgt
tgcattcacc aaacagcatt ccatccatca acatggatgt 1980 aaagcctaac
gaggaacaaa tgtcgatgaa gtttggtgat gataggcagg acagagatgg 2040
attctcacta atgggaggac cgatgaactt catgggagga ttcggagcct atcccattgg
2100 agaaattgct cggtttagca ccgagcaatt ctcagcacca tactcaacca
gtggcacagt 2160 ttcactcact cttggcctac cacataacga aaacctctca
atgtctgcaa cacaccacag 2220 tttccttcca attccaacac aaaacatcca
aattggaagt gaaccaaatc atgagtttgg 2280 tagcttaaac acaccaacat
cagctcactc aacatcaagc gtctatgaaa ccttcaacat 2340 tcagaacaga
aagaggttcg ccgcaccctt gttaccagat tttgttgcct gatcacaaaa 2400
acaaaaacag gttttggcaa cagacaaact tctgtcgcta aacaaggaca tgatttagcg
2460 acagataact tcagtcgcta acttagcgac tgaaaacttc tgtcgctaag
catgaacatg 2520 tattagcgac atacagtatg caactgtatg tcactaaaca
agaacatgat gaattagtga 2580 cggacaactt ctgtcgctaa acaacaaaaa
aaaatccatg ttttagtata ttgtttctca 2640 ttctatcata tcatggtagt
gtaaagaatc aagaaacaag ttttacatag taacagtctt 2700 tatacattgg
agatgaagaa ccatttaagt tcttcaaaat agatagattt tctaggttac 2760
ttctacaaga tatatatatg gttgagggtt tgtatattaa ttttgggatt gttatattgg
2820 atgtggaaaa aaagtagtta ttttgggtgg tataaataaa ataatactcc
atccatttta 2880 gccaaaaaaa aaaaaaaa 2898
[0062] The mRNA of StBEL29 described above is derived from StBEL29,
having a nucleotide sequence of SEQ ID NO:5, as follows, where the
upstream sequence is shown in italics, introns are shown in regular
font, and exons are shown in bold:
TABLE-US-00005 tgagaagaaa acccaaagaa acttatgatt tataataaat
tattagaaat ttctatggat 60 ataaaatggt aaaaagtaag ttttattaaa
tataaaaata tgtttttttt aatggaataa 120 aaagcaaaaa aaaatcacat
aaattagaat aaagatcgga gaaagtaaat tataaataaa 180 gacaagatga
aaaacaaggc gataatgtaa atcatactaa tcaatcgtta tacatattaa 240
aaaatatcca gcgttacaac aacaaattta acaatataat ataataaaat ttaactaaaa
300 atcaaaataa aatgacattt atcataacaa taattaacaa ccatccaaat
atgatgtatg 360 gataaaaggt gaagagtatt agtatctttt gtttaaatct
tatatattaa aattataaat 420 ttaattatta ttttaaaaat tcttatataa
attttaaatt ctgaatttgt ccgacggcta 480 atctaaagtc aaaagtaaat
tttcataaat gtaggtccta aattttttcc cacaattatc 540 ttcttccaag
ttgccaacac aaatcaataa tgacaatagg gccctctccc ctatctcttc 600
aaccctacct ctctttttct ttctttatca cttcaagttc atatcatatt tcatactctc
660 tcattttctt ctggtctccg ttgtaattta tatgatatat tttttaatat
ttaaaataat 720 ttaattttaa attttttata ctctttaaaa aattattata
atcataagtt ataaaaaaaa 780 ttaacttttt tttattcagt caaatactat
catataaatt aaaaaagaaa aagtatatgt 840 taaatcctta taattattat
tgttaaagaa gaaaaaaggg aggttagtgg aagtggacgt 900 tacctcgttt
ttcatctgtc tgttttttct gacacacctt tgatctttga tgatggatac 960
gtcgctccgt tcatatttag gtgatactat attaatttca agagttaaat aatgataaat
1020 cacctaagac cgctaatgtt ccatctaatt caagaacaag cccttctcaa
tgtcttgcct 1080 ttcgcatgtg ttttctttga aattggaatt ccaaccaagt
tcccttccca aagcgggaac 1140 aagttggtgc gaccgattaa agaagaagga
caaagagtta aataatgaaa ttataattat 1200 tttatattaa ttattataat
ttataatatt ttttaaaaac taaatgttct aatttaaagg 1260 caaagtccaa
atatttattt tataaatttt gaagcataat tgggttttga ttaattattt 1320
atatcaaatt aaatttattt taatacaaat acataattta agacaaagct attgagttaa
1380 agttatgtca aattaaatcc gtaactttat aagctcaagg ggagaaagag
agaaggattg 1440 ttcattcctt ataacgagtc tagagatctc atcctttatc
gatgtaaggt tctttccatt 1500 catcactccc ttgcgttaga accttttttt
tttagactgg agcgtgcaca ttcatggacc 1560 attcttccca ttcgtcaatc
cctcgtgtta gaatttttat ttctcgaact agagtgtgtg 1620 cattaacaga
taccagatac cgatattttc accctcattc aagccgtctc tggaagagct 1680
atattggatg agcctgactt tgataccata tcaaattaac tcttcaacct aattcataca
1740 tcaaaagcta gctcgcctta taagaagtct ttccattcgt cactccctcg
tgttacaact 1800 tacaagacta gctcaataaa aaattatcgt ccaaatttta
taagaagtcc attcatcaat 1860 agcacctttc ctatttgtat ttgcacttaa
aaaaaaaaag gtgacttttg aaatttgaat 1920 tatgccacat aaattatcct
tcggtatagc ccaatgattt gaccttggta ctttcatatt 1980 ggaggtctca
aatttgaaat tccttaccag taaaaataaa aaatttacct tcctgaatcg 2040
aacttatcgc gccagacttc cttagacaca caaattagaa taaaaaaagt atattttatt
2100 tttatatata agcaaaaaca cacactaact cacattcaca catccacatc
tttctttctt 2160 tctttctcct ctctctctct ctaaaaagtt gagtactttt
attagctctc atcacttcac 2220 acagaagaag atggtatttt tatttctttc
tgctgatggc tgcatcaaat gatttgaaaa 2280 gctgagtcaa atcagaagaa
gaaaaagaaa gttataataa taataataat aatatcaaaa 2340 atattatttt
caggtatggt acttctttac tcattaacaa tgtaaatata gaatttgaag 2400
tttacgagct agttttctct tcttttttat tttgactagc agaaacagag tcagagtcag
2460 aatttgaagt ttataagtct tgaattctga ttttgtttga gttcttgagt
tctgaattga 2520 taatttatac atgttgaatg aattttgtaa gtatactttg
aaacaaatct attgagttcg 2580 attgaattca taaccgacac tttagttccg
ccacttttca gaggcggatc cagaatatga 2640 aggttatgac ttatgagtat
tgtaaccttt tgagttactg aattctaaat taattttata 2700 agtgagtaaa
tacaaaattt gaaacaaaat tagctattga gttcagttga attcgtataa 2760
ccgacactct agctttgtca ctgctcaaag actgatctag aatttgaagt ttatgagttt
2820 tgaattctaa attgataatt tctacatgtt agatggaatt tttaagataa
atataaatta 2880 aaattattga gttcgatcga attcgatttg tgttttcctt
tttcttcacc ttatttatca 2940 agagaaatta ttttaatttt tttttatcat
tactgattca taaatctata tagatatata 3000 tagatggata cattagagtt
cctaaaaaat gttataaaga gtattttgtt tttccctttc 3060 ttgatttttt
ttcgaaacta agatttcaat tttatcattt ctgaatttta taacaacgat 3120
tatcatagaa ttctaaattt actagttata catatttatt taacgaatta ttaacataaa
3180 tgcattattt gaataaaatt tattagattt gaccgaactc gtatatgaac
tttctctctc 3240 atgtaatttc agccgtaact gtgtgtattt cctttctctc
tctaaaaaat ctgttatggt 3300 gttctgtgtc actctaacaa aaaataaatt
attcttcatt tcttccattg tcagattata 3360 atacaccacg tgcacttaca
aattttgtga aaaccatatt ttaatttaca aatccttttc 3420 ataattcttt
tttttaatca agaaaattaa atttaattac attaagtatt ttttacaata 3480
atattaacat attactaaat aaattttcag gtttatttta ccatttttgt gattttgaag
3540 tgttacaaag tgtgaatggg ttggactttt tggacctcag ctaggtagct
tcttgtcttc 3600 aacacaaaag gtacatataa aaattacaat aaaattataa
ccacttttac ttaggaattt 3660 taccatattt aggtaaaaaa aataaatcac
tcgcctagtc gtaataatca gtagcagagc 3720 tagaggaacg aaaggcttca
tctgaatctt ctttatctga aatcatactg tatataaggt 3780 caaaattcat
tttttatgaa cacttttgat gaaaatcatg tctctgccac taaagtcgta 3840
atttgtaggc atcaacaata tgtatatata tatatatata gtgattattg attatatacg
3900 gtatatattg gttaaaagtt ttgaaaaata aggattaaag ttaatttcca
ttgtgtgttt 3960 tcttgggatc aggggcggag ctagataagt gtaaaaaggg
tttatctaga cttcttccga 4020 ccaaaaatta tacttatata catatacata
gtagatactg aatcccttgc ttttttcgta 4080 tatgtacttc cgcatatttt
aaatttcttt aatgaaaatt ctgactcata tactgtttga 4140 atgtttttgt
atttaatata tgtatgtttt gcctttttat tttggaaaaa atgatatatg 4200
tggacttact tgacttgact ttaacttatt tttttttatt tttcagatta gttggtgtta
4260 tttgtttata gtggagaaaa aataaattaa aaaagaagag aaaatggcat
cttattttca 4320 tggaaattca gaaatacaag aaggaaatga tggattacaa
actctaatac taatgaatcc 4380 tggatatgtt ggattttctg aaacacaaca
tcaccacgcg ccgccgccgc caggtggcag 4440 cagcaacaac atagttttct
tcaactccaa tcctcttgga aattcaataa acttatctca 4500 cgcgccacca
cctccgccac cgccacaaca acatttcgtc ggtatacctc tcgccaccgc 4560
cgccttcacc gccccatccc aagactccgg taacaacaac aacaacgagt caatctccgc
4620 ccttcacggc ttcctagctc gatcgtctca gtacgggttt tacaacccgg
ctaacgacat 4680 cacggcggcg cgtgaggtca cacgcgctca tcatcagcag
cagcaagggc tttcacttag 4740 cctgtcctca tcccagcagc ctgggtttgg
gaacttcacg gcggcgcgtg agattgtttc 4800 ttcgcctacg cgttcggctt
cggcttccgg gatacaacaa caacaacagc aacaacaaag 4860 tattagtagt
gtgcctttga gttctaagta catgaaggct gcacaagagc tacttgatga 4920
agttgtaaat gttggaaaat caatgagaag tactaatagt actgaagttg ttgttaataa
4980 tgatgtcaag aaatcgaaga ttatgaccga tatggatgga cagatagatg
gaggagcaga 5040 caaagacgga actccaacaa ctgagctaag taccgcagag
aggcaagaaa ttcaaatgaa 5100 gaaagcaaaa cttgttaaca tgcttgacga
ggtaaccttg ttgtcttttt ctcagtaatg 5160 ttgttgcatt cgtgtcagat
cagagtctta aaattagtca atagaagaaa cttcatttcc 5220 tcgagtacgt
gtaattgtgg ccttttcgac ttccaactag tatttacaat agtgcactct 5280
acattgataa gcttgacgac aagtaggcaa agcgatggcc ttgttggttg ttatagtttt
5340 ttggttatgt tgctcggact ctgcaaaatt attgtcatac tcaagtcaga
ttctccaaaa 5400 tgcactattt ttggagtatc cgacttgcag tctgacattt
attttttccg aagagtctga 5460 gcaacatagg ttttcttggc tttccaagat
agtaagagaa tggtctctat caaaaaaagt 5520 tacatcatat cattactgaa
aataagagca aaaaagtatc tgtcaaatga taaagaccag 5580 aacttcaaaa
ctgttacttt cgtcagggca ctgtcttgac aattgtaaac aaaaaatgaa 5640
agaatttttc gaaaataatt tcttcgaaat ctttgatcta aagctaaata tcggttcgat
5700 tttgggtgtt gttatatagg tggagcagag gtatagacat tatcatcacc
aaatgcagtc 5760 agtgatacac tggttggagc aagctgctgg tattggatca
gcaagaacat atacagcatt 5820 ggctttgcag acgatttcga agcaatttag
gtgtcttaag gacgcgataa ttggtcaaat 5880 acgatcagca ggcaagacgt
taggcgaaga agatagtttg ggagggaaga ttgaaggttc 5940 aaggcttaaa
tttgttgaca atcagctaag acagcaaagg gctttgcaac aattgggaat 6000
gatccagcat aatgcttgga gacctcagag aggattgccc gaacgagctg tttctgttct
6060 tcgcgcttgg ctttttgaac atttcctcca tccgtaagca cgaaacaacc
ctttttcatc 6120 agctatgttg ctcggacttt tcaaaaacgt tgtcgcacca
gtgttggatc ctcgcagaat 6180 gcattgattt tttgaggatc cgacacatac
ctgacgatat ttttgaagag tctgaacaac 6240 atagcttagt taaaagtact
gtattttgat atattgtggc aatttgtttt gtatagctat 6300 cccaaggatt
cagacaaaat gatgctagca aaacaaacag ggctaactag gagtcaggtc 6360
agtgatatct gataacaaca ttgtcatttt tgattctcga gttgatttct cagatggtca
6420 cttaactgta gttattatat cagaaagtcg ccttacttca acaaagagag
tgacattctg 6480 agataataac tgtgagttga gtgaccatct gagaaatcaa
ctcttggatt ctccgttttt 6540 ggtttttact aagttttgtt tttggacaat
tcaggtgtcg aattggttca tcaatgctcg 6600 agttcgtctt tggaagccaa
tggtggaaga gatgtacttg gaagagataa aagaacagaa 6660 cggattgggt
caagaaaaga cgagcaaatt aggcgaacag aacgaagatt caacaacatc 6720
aagatccatt gctacacaag acaaaagccc tggttcagat agccaaaaca agagttttgt
6780 ctcaaaacag gacaatcatt tgccccaaca caaccctgct tcaccaatgc
cgatgtccaa 6840 caccacttcc atacctccta tcggtatgaa catccgtaat
cagtctgctg gtttcaacct 6900 cattggatca ccagagatcg aaagcatcaa
cattactcaa gggagtccaa agaaaccaag 6960 gaacaacgag atgttgcatt
caccaaacag cattccatcc atcaacattg atgtaaagcc 7020 taacgagcaa
caaatgtcga tgaagtttgg tgatgatagg caagacagag atggattctc 7080
actaatggga ggaccgatga acttcatggg aggattcgga gcctatccca ttggagaaat
7140 tgctcggttt agcaccgagc aattctcagc accatactca accagtggca
cagtttcact 7200 cactcttggc ctaccacata acgaaaacct ctcaatgtca
gcaacacacc acagtttcct 7260 tccaattcca acacaaaaca tccaaattgg
aagtgaacca aatcatgagt ttggtagctt 7320 aaacacacca acatcagctc
actcaacatc aagcgtctac gaaaatttca acattcagaa 7380 cagaaagagg
ttcgccgcac ccttgttacc agattttgtt gcctgatcac aaaaacaaaa 7440
acaggattta gcgacagaca aacttctgtc gctaaacaag aacatgattt agcgacagat
7500
aacttcagtc gctaacttag cgactgaaaa cttctgtcgc taaacatgaa catgtattag
7560 cgacatacag tatacaactg tatgtcgcta aacaagaaca tgatgaatta
gtgacggaca 7620 acttctgtcg ctaaacaaca aaaaaagatc catgttttag
tatattgttt ctcattctat 7680 catatcatgg tagtgtaaag aatcaagaaa
caagttttac atagttacat agtctttata 7740 cattggagat gaagaaccat
ttaagttctt caaaatagat agattttcta ggttacttct 7800 agaagatata
tatatggttg agggtttgta tattaatttt gggattgtta tattggatgt 7860
ggaaaaaaag tagttatttt gggtggtata aataaaataa tactccatcc attttagcca
7920 a 7921
[0063] In one embodiment, the second nucleic acid molecule
comprising a nucleotide sequence configured to silence or reduce
expression of StBEL29 has a nucleotide sequence of SEQ ID NO:6, as
follows:
TABLE-US-00006 gtgttatttg tttattgtgg agaaaaaata aattaaaaag
gaagaaaaaa tggcatctta 60 ttttcatgga aattcagaaa tacatgaagg
aaatgatgga ttacaaactc taatactaat 120 gaatcctgga tatgttggat
tttctgaaac acaacatcac cacgcgccac cgccgccgcc 180 gccaggtggc
agcagcagca acatagtttt cttcaactcc aatcctattg gaaattcaat 240
gaacttatct cacgcgccac cacctcctcc accgcctcaa caacaattca tcggtatacc
300 cctcgccacc gccgccttca ccgccccatc ccaagactcc ggtaacaaca
acaacaacga 360 gtcaatctcc gcccttcacg gcttcctagc tcgatcgtct
cagtacgggt tttacaaccc 420 ggcaaacgac ctcacggcgg cgcgtgacgt
cacacgcgct catcatcatc atcagcagcc 480 aagggctttc acttacctgt
cctcgtccca gcagccgggg tttgggaact tcacggcggc 540 gcgtgagctt
gtttcttcgc cttcgggttc ggcttcagct tcagggatac aacaacaaca 600
acagcaacaa cagagtatta gtagtgtgcc tttgagttct aagtacatga aggctgcaca
660 agagctactt gatgaagttg taaatgttgg aaaatcaatg aaaagtacta
atagtactga 720 tgttgttgtt aataatgatg tcaagaaatc gaagaatatg
ggcgatatgg acggacagtt 780 agacggagtt ggagcagac 799
[0064] Other nucleic acid molecules may also be used as the second
nucleic acid molecule comprising a nucleotide sequence configured
to silence or reduce expression of StBEL29. For example, in certain
embodiments, the second nucleic acid molecule has a nucleotide
sequence that is at least about 95%, 96%, 97%, 98%, or 99%
identical to the nucleotide sequence of SEQ ID NO:6.
[0065] By way of other examples, the second nucleic acid molecule
has a nucleotide sequence that is at least about is at least 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% identical to the
nucleotide sequence of SEQ ID NO:6.
[0066] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous bp of SEQ
ID NO:6.
[0067] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous by of SEQ
ID NO:5 which are less than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10%, 5%, or 1% identical to any portion of SEQ
ID NO:8, where the portion of SEQ ID NO:8 has the same length as
the first nucleic acid molecule.
[0068] According to this embodiment, one can silence or reduce
expression of StBEL29 without silencing or reducing the expression
of StBEL5.
[0069] Nucleic acid molecules capable of silencing or reducing
expression of StBEL29, using any of the methods described infra are
contemplated.
[0070] In one embodiment, the first nucleic acid molecule comprises
a nucleotide sequence configured to silence or reduce expression of
both StBEL11 and StBEL29. This is possible because of the
similarity in sequence between StBEL11 and StBEL29.
[0071] In yet another embodiment of this aspect of the present
invention, the nucleic acid construct further comprises a further
nucleic acid molecule comprising a nucleotide sequence configured
to enhance the expression of StBEL5 and variants thereof. This
additional nucleic acid molecule may be included in the nucleic
acid construct of this aspect of the present invention in addition
to the second nucleic acid molecule (described supra) or in place
of the second nucleic acid molecule.
[0072] The mRNA of StBEL5 has a nucleotide sequence of SEQ ID NO:7,
as follows, where 5' UTR and 3' UTR sequences are shown in italics,
and the CDS is shown in regular font:
TABLE-US-00007 catgcagaga taaaaatata gatcagtctg acaagaaggc
aacttctcaa agcttagaga 60 gctaccaccc gaagatagac agttagttac
atgtactgtt atagataaaa ggagaaatcc 120 gaagaagaaa gaattttttt
tgcagatatg tactatcaag gaacctcgga taatactaat 180 atacaagctg
atcatcaaca acgtcataat catgggaata gtaataataa taatattcag 240
acactttatt tgatgaaccc taacaattat atgcaaggct acactacttc tgacacacag
300 cagcagcagc agttactttt cctgaattct tcaccagcag caagcaacgc
gctttgccat 360 gcgaatatac aacacgcgcc gctgcaacag cagcactttg
tcggtgtgcc tcttccggca 420 gtaagtttgc acgatcagat caatcatcat
ggacttttac agcgcatgtg gaacaaccaa 480 gatcaatctc agcaggtgat
agtaccatcg tcgacggggg tttctgccac gtcatgtggc 540 gggatcacca
cggacttggc gtctcaattg gcgtttcaga ggccgattcc gacaccacaa 600
caccgacagc agcaacaaca gcaaggcggt ctatctctaa gcctttctcc tcagctacaa
660 cagcaaatta gtttcaataa caatatttca tcctcatcac caaggacaaa
taatgttact 720 attaggggaa cattagatgg aagttctagc aacatggttt
taggctctaa gtatctgaaa 780 gctgcacaag agcttcttga tgaagttgtt
aatattgttg gaaaaagcat caaaggagat 840 gatcaaaaga aggataattc
aatgaataaa gaatcaatgc ctttggctag tgatgtcaac 900 actaatagtt
ctggtggtgg tgaaagtagc agcaggcaga aaaatgaagt tgctgttgag 960
cttacaactg ctcaaagaca agaacttcaa atgaaaaaag ccaagcttct tgccatgctt
1020 gaagaggtgg agcaaaggta cagacagtac catcaccaaa tgcaaataat
tgtattatca 1080 tttgagcaag tagcaggaat tggatcagcc aaatcataca
ctcaattagc tttgcatgca 1140 atttcgaagc aattcagatg cctaaaggat
gcaattgctg agcaagtaaa ggcgacgagc 1200 aagagtttag gtgaagagga
aggcttggga gggaaaatcg aaggctcaag actcaaattt 1260 gtggaccatc
atctaaggca acaacgcgcg ctgcaacaga taggaatgat gcaaccaaat 1320
gcttggagac cccaaagagg tttacctgaa agagctgtct ctgtccttcg tgcttggctt
1380 ttcgagcatt ttcttcatcc ttacccaaag gattcagaca aaatcatgct
tgctaagcaa 1440 acggggctaa caaggagcca ggtgtctaac tggttcataa
atgctcgagt tcgattatgg 1500 aagccaatgg tagaagaaat gtacttggaa
gaagtgaaga atcaagaaca aaacagtact 1560 aatacttcag gagataacaa
aaacaaagag accaatataa gtgctccaaa tgaagagaaa 1620 catccaatta
ttactagcag cttattacaa gatggtatta ctactactca agcagaaatt 1680
tctacctcaa ctatttcaac ttcccctact gcaggtgctt cacttcatca tgctcacaat
1740 ttctccttcc ttggttcatt caacatggat aatactacta ctactgttga
tcatattgaa 1800 aacaacgcga aaaagcaaag aaatgacatg cacaagtttt
ctccaagtag tattctttca 1860 tctgttgaca tggaagccaa agctagagaa
tcatcaaata aagggtttac taatccttta 1920 atggcagcat acgcgatggg
agattttgga aggtttgatc ctcatgatca acaaatgacc 1980 gcgaattttc
atggaaataa tggtgtctct cttactttag gacttcctcc ttctgaaaac 2040
ctagccatgc cagtgagcca acaaaattac ctttctaatg acttgggaag taggtctgaa
2100 atggggagtc attacaatag aatgggatat gaaaacattg attttcagag
tgggaataag 2160 cgatttccga ctcaactatt accagatttt gttacaggta
atctaggaac atgaatacca 2220 gaaagtctcg tattgatagc tgaaaagata
aaaggaagtt agggatactc ttatattgtg 2280 tgaggccttc tggcccaagt
cggaggaccc aatttgatac aacctatcat aggagaaaag 2340 aagtggagac
taaattaaag taacaaaatt ttaaagcaca ctttctagta tatatacttc 2400
ttttttttat agtatagaaa agaagagatt ttgtgcttta gtgtatagat agagtctact
2460 tagtataggt tatacttcta gttccttgag aagattgata caactagtag
tatttttttt 2520 cttttgggtt ggcttggagt actattttaa gttattggaa
actagctata gtaaatgttg 2580 taaagttgtg atattgttcc tctcaatttg
catataattt gaaatatttt gtacctacta 2640 gctagtctct aaattatgtt
tccattgctt gtaattgcaa ttttatttga attttgtgct 2700 atcattatta
gattagcaaa aaaaaaaaaa aaaaa 2735
[0073] The mRNA of StBEL5 described above is derived from StBEL5,
having a nucleotide sequence of SEQ ID NO:8, as follows, which
includes UTR, exons, and intronic sequence:
TABLE-US-00008 gtaggtacaa aatatttcaa attatatgca aattgagagg
aacaatatca caactttaca 60 acatatacta tagctagttt ccatattaac
ttaaaatagt actccaagcc aacccagaaa 120 gaaaaaaaat actactagtt
gtatcaatct tctcaaggaa ctagaagtat aacctatact 180 aagtagactc
tatctataca ctaaagcaca aaatctcttc ttttctatac tataaaaaaa 240
agaagtatat atactagaaa gtgtgcttta aaattttgtt actttaattt tgtctccact
300 tcttttctcc tatgataggt tgtatcaaat tgggtcctcc gacttgggcc
agaaggcctc 360 acacaatata agagtatccc taacttcctt ttatcttttc
agctatcaat acgagacttt 420 ctggtattca tgttcctaga ttacctgtaa
caaaatctgg taatagttga gtcggaaatc 480 gcttattccc actctgaaaa
tcaatgtttt catatcccat tctattgtaa tgactcccca 540 tttcaggcct
acttcccaag tcattagaaa ggtaattttg ttggctcact ggcatggcta 600
ggttttcaga aggaggaagt cctaaagtaa gagagacacc attatttcca tgaaaattcg
660 cggtgatttg ttgatcatga ggatcaaacc ttccaaaatc tcccatcgcg
tatgctgcca 720 ttaaaggatt agtaaaccct ttatttgatg attctctagc
tttggcttcc atgtcaacag 780 atgaaagaat actacttgga gaaaacttgt
gcatgtcatt tctttgcttt ttcgcgttgt 840 tttcaatatg atcaacagta
gtagtagtag tattatccat gttgaatgaa ccaaggaagg 900 agaaattgtg
agcatgatga agtgaagcac ctgcagtagg ggaagttgaa atagttgagg 960
tagaaatttc tgcttgagta gtagtaatac catcttgtaa taagctgcta gtaataattg
1020 gatgtttctc ttcatttgga gcctctttgt ttttgttatc tcctgaagta
ttagtactgt 1080 tttgttcttg attcttcact tcttccaagt acatttcttc
taccattggc ttccataatc 1140 gaactcgagc atttatgaac cagttagaga
cctgcatttt catatatatt aagaattttt 1200 ttataaagaa aagaaaggaa
ttaatccaag aattatagta aaatgtgtgt ctaagaacct 1260 ggctccttgt
tagccccgtt tgcttagcaa gcatgatttt gtctgaatcc tttgggtaac 1320
tgcaatataa aaatatatta agaaaaaaaa aattatagtt aaaaacatac tcctatatta
1380 gagaagaatg ggcgaattca gaatttagaa taataatgtg atcttattat
atacatgaac 1440 atattttatt ttttgtgtgt atgcatatat agtttgagtt
aaaagtaagt gtctttttaa 1500 ccgtccaaac gaaagtttca aaataactgg
cgttgctcta aaaatcactt aatttgttcc 1560 taatggatgg acatgttaaa
accatataaa agacacttag taaaatatag gacgacaggc 1620 gtatatatga
cgcaaaaatt ggttagaata atcaattttc tatctactac tccgtagata 1680
tttttcacat tgttaaattt ttcaaagaaa taaatagttt aggagtactc acggatgaag
1740 aaaatgctcg aaaagccaag cacgaaggac agagacagct ctttcaggta
aacctctttg 1800 gggtctccaa gcatttggtt gcatcattcc tagctgttgc
agcgcgcgtt gttgccttag 1860 atgatggtcc acaaatttga gtcttgagcc
ttcgattttc cctactaagc cttcctcttc 1920 acctaaactc ttgctcgtcg
cctttacttg ctcagcaatt gcatccttta ggcatctgaa 1980 ttgcttcgaa
attgcatgca aagctaattg agtgtatgat ttggctgaac caattcctgc 2040
tacttgctca aatgatgata caattatttg catttggtga tggtactgtc tgtacctttg
2100 ctccacctga acaaaaaaaa gggagtaata ttaaactttt accagtctgt
cgttttacaa 2160 catgaagtta tcttatgttg gctactattg aaattaaaga
attttatttc agttaaaaga 2220 tcatatatat atatatatat atattccaag
tgagaaataa attgagtagt atattttgca 2280 aaattttgta aaccaacgaa
tttttgagag tcattagatt gaggacacat ctgagtggac 2340 attatgcgtg
gtgtaaaaaa ggtgaataag agatagtggt ttgaattttg gtgcagcgga 2400
agacatttca ggttcgtagc taactttggt gtattatctt tatagcttta gttggacccg
2460 cagaagaaaa tttaagagcc acacattgtc agtttgtttt aatatcaacg
tacgtgattg 2520 gtctcttgtt cttcaactaa ttaacaaaac ctgtacattt
catttaccaa ctactattgt 2580 tgcaaacata tataaatcaa cagtttcatc
cattcaattt tttatgagaa aaattacagt 2640 tttgaatcat ttgaaaataa
aattttaaat atatatgtcg aattcagtag ttttagtgtt 2700 aagaatccga
aattcataga ctcaaaattc aggatcatac ctcttcaagc atggcaagaa 2760
gcttggcttt tttcatttga agttcttgtc tttgagcagt tgtaagctca atagcaactt
2820 catttttctg cctgctgcta ctttcaccac caccaccacc accagaacta
ttagtgttga 2880 catcactagc caaaggcatt gaattatcct tcttttgatc
atctcctttg atgctttttc 2940 caacaatatt aacaacttca tcaagaagct
cttgtgcagc tttcagatac ttagagccta 3000 aaaccatgtt gctagaactt
ccatctaatg ttcctctaat agtaacatta tttgtccttg 3060 gtgatgagga
tgaaatattg ttattgaaac taatttgctg ttgttgctga ggagaaaggc 3120
ttagagatag accgccttgc tgttgttgct gctgctgtcg gtgttgtggt gtcggaatcg
3180 gcctctgaaa cgccaattga gacgccaagt ccgtggtgat cccgccacat
gacgtggcag 3240 aaacccccgt cgacgatggt actatcacct gctgagattg
atcttggttg ttccacatac 3300 gctgtaaaag tccatgatga ttgatctgat
cgtgcaaact tactgccgga agaggcacac 3360 cgacaaagtg ctgctgttgc
agcggcgcgt gttgtatatt cgcatggcaa agcgcgttgc 3420 ttcctgctgg
tgaagaattc aggaaaagta actgctgctg ctgctgtgtg tcagaagtag 3480
tgtagccttg catataattg ttagggttca tcaaataaag cgtctgaata ttattattat
3540 tactactatt cccatgatta tgatgttgtt gatgatcagc ttgtatatta
ttatccgagg 3600 ttccttgata gtacatatct gcaaaaaaaa atctttcttc
ttcgaatttc tccttttatc 3660 tacaacagta cctgtaaaca gaaagtaaca
aaggagaaaa ggcttcaaat aagtccacac 3720 aaacattttt ataagtaaac
ggaagggaat tctttatagt gaaaaattaa attttgttta 3780 cagagatctt
caactataaa taaaaaaaac aggaaaatga tataaaagaa agagaaagag 3840
atgaaaggag ctttagcaaa aaaatcagtc actcacacat acacacatgt aactaactgt
3900 ctatcttcgg gtggtagctc tctaagcttt gagaagttgc cttcttgtca
gactgatcta 3960 tatttttctc tctgcattct catctcttca accacaaaaa
ggaaatatga ataaa 4015
[0074] In one embodiment, the further nucleic acid molecule
comprises a nucleotide sequence of SEQ ID NO:7 or a nucleic acid
molecule that is at least about 95%, 96%, 97%, 98%, or 99%
identical to the nucleotide sequence of SEQ ID NO:8. Alternatively,
the further nucleic acid molecule comprises a nucleotide sequence
capable of expressing active and/or functional StBEL5 to enhance
the expression of StBEL5. A description of enhancing the expression
of StBEL5 is provided infra.
[0075] Another aspect of the present invention is directed to a
nucleic acid construct comprising a first nucleic acid molecule
comprising a nucleotide sequence configured to silence or reduce
expression of StBEL29 and variants thereof; a 5' DNA promoter
sequence; and a 3' terminator sequence, where the first nucleic
acid molecule, the promoter sequence, and the terminator sequence
are operatively coupled to permit transcription of the first
nucleic acid molecule.
[0076] According to one embodiment, the nucleic acid construct of
the present invention comprises DNA heterologous to the first
nucleic acid molecule. In one particular embodiment, the DNA
heterologous to the first nucleic acid molecule is the 5' DNA
promoter sequence.
[0077] In one embodiment, the first nucleic acid molecule
comprising a nucleotide sequence configured to silence or reduce
expression of StBEL29 has a nucleotide sequence of SEQ ID NO:6.
[0078] Other nucleic acid molecules may also be used as the first
nucleic acid molecule comprising a nucleotide sequence configured
to silence or reduce expression of StBEL29. For example, in certain
embodiments, the second nucleic acid molecule has a nucleotide
sequence that is at least about 95%, 96%, 97%, 98%, or 99%
identical to the nucleotide sequence of SEQ ID NO:6.
[0079] By way of other examples, the first nucleic acid molecule
has a nucleotide sequence that is at least about 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, or 94% identical to the nucleotide
sequence of SEQ ID NO:6.
[0080] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous bp of SEQ
ID NO:6.
[0081] In certain embodiments, the first nucleic acid molecule
comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous by of SEQ
ID NO:5 which are less than 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10%, 5%, or 1% identical to any portion of SEQ
ID NO:8, where the portion of SEQ ID NO:8 has the same length as
the first nucleic acid molecule.
[0082] According to this embodiment, one can silence or reduce
expression of StBEL29 without silencing or reducing the expression
of StBEL5.
[0083] In yet another embodiment of this aspect of the present
invention, the nucleic acid construct further comprises a further
nucleic acid molecule comprising a nucleotide sequence configured
to enhance the expression of StBEL5 and variants thereof.
[0084] In one embodiment, the further nucleic acid molecule
comprises a nucleotide sequence of SEQ ID NO:7 or a nucleic acid
molecule that is at least about 95%, 96%, 97%, 98%, or 99%
identical to the nucleotide sequence of SEQ ID NO:5. Alternatively,
the further nucleic acid molecule comprises a nucleotide sequence
capable of expressing active and/or functional StBEL5 to enhance
the expression of StBEL5. A description of enhancing the expression
of StBEL5 is provided infra.
[0085] Methods for identifying nucleic acid molecules capable of
silencing or reducing expression of genes and/or related RNA
molecules are well known in the art and are discussed in more
detail infra.
[0086] As discussed supra, the nucleic acid constructs of the
present invention comprise, in one embodiment, one or more nucleic
acid molecules comprising a nucleotide sequence configured to
silence or reduce expression of mRNA molecules and/or genes. In
some embodiments, the nucleic acid constructs include a nucleic
acid molecule comprising a nucleotide sequence configured to
enhance expression of mRNA molecules and/or genes.
[0087] General strategies for silencing or reducing expression and
enhancing expression are known in the art. Up-regulation,
down-regulation, ectopic expression, gene editing, or gene
silencing are well known.
[0088] Silencing or reducing gene expression means the interruption
or suppression of the expression of a gene at the level of
transcription or translation. In the present invention, silencing
of StBEL gene expression may be carried out, according to one
embodiment, by a nucleic acid molecule of the construct containing
a dominant mutation and encoding a non-functional StBEL, resulting
in suppression or interference of endogenous mRNA encoding the
StBEL or variant thereof.
[0089] In another embodiment, the nucleic acid construct results in
interference of StBEL gene expression by sense or co-suppression in
which the nucleic acid molecule of the construct is in a sense
(5'->3') orientation. Co-suppression has been observed and
reported in many plant species and may be subject to a transgene
dosage effect or, in another model, an interaction of endogenous
and transgene transcripts that results in aberrant mRNAs (Senior,
"Uses of Plant Gene Silencing," Biotechnology and Genetic
Engineering Reviews 15:79-119 (1998); Waterhouse et al., "Exploring
Plant Genomes by RNA-Induced Gene Silencing," Nature Review:
Genetics 4:29-38 (2003), which are hereby incorporated by reference
in their entirety). A construct with the nucleic acid molecule in
the sense orientation may also give sequence specificity to RNA
silencing when inserted into a vector along with a construct of
both sense and antisense nucleic acid orientations as described
infra (Wesley et al., "Construct Design for Efficient, Effective
and High-Throughput Gene Silencing in Plants," Plant Journal
27(6):581-590 (2001), which is hereby incorporated by reference in
its entirety).
[0090] In yet another embodiment, the nucleic acid construct
results in interference of StBEL gene expression by the use of
antisense suppression in which the nucleic acid molecule of the
construct is an antisense (3'.fwdarw.5') orientation. The use of
antisense RNA to down-regulate the expression of specific plant
genes is well known (van der Krol et al., "An Anti-sense Chalcone
Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation,"
Nature 333:866-869 (1988) and Smith et al., "Antisense RNA
Inhibition of Polygalacturonase Gene Expression in Transgenic
Tomatoes," Nature 334:724-726 (1988), which are hereby incorporated
by reference in their entirety). Antisense nucleic acids are DNA or
RNA molecules that are complementary to at least a portion of a
specific mRNA molecule (Weintraub, "Antisense RNA and DNA,"
Scientific American 262:40 (1990), which is hereby incorporated by
reference in its entirety). In the target cell, the antisense
nucleic acids hybridize to a target nucleic acid and interfere with
transcription, and/or RNA processing, transport, translation,
and/or stability. The overall effect of such interference with the
target nucleic acid function is the disruption of protein
expression (Baulcombe, "Mechanisms of Pathogen-Derived Resistance
to Viruses in Transgenic Plants," Plant Cell 8:1833-44 (1996);
Dougherty, et al., "Transgenes and Gene Suppression: Telling us
Something New?," Current Opinion in Cell Biology 7:399-05 (1995);
Lomonossoff, "Pathogen-Derived Resistance to Plant Viruses," Ann.
Rev. Phytopathol. 33:323-43 (1995), which are hereby incorporated
by reference in their entirety). Accordingly, one embodiment
involves a nucleic acid construct which contains the StBEL gene
encoding nucleic acid molecule being inserted into the construct in
antisense orientation.
[0091] Interfering with endogenous StBEL gene expression may
involve an RNA-based form of gene-silencing known as RNA
interference ("RNAi") (also known as siRNA for short, interfering
RNAs). RNAi is a form of post-transcriptional gene silencing
("PTGS"). PTGS is the silencing of an endogenous gene caused by the
introduction of a homologous double-stranded RNA ("dsRNA"),
transgene, or virus. In PTGS, the transcript of the silenced gene
is synthesized, but does not accumulate because it is degraded.
RNAi is a specific form of PTGS, in which the gene silencing is
induced by the direct introduction of dsRNA. Numerous reports have
been published on critical advances in the understanding of the
biochemistry and genetics of both gene silencing and RNAi (Matzke
et al., "RNA-Based Silencing Strategies in Plants," Curr. Opin.
Genet. Dev. 11(2):221-227 (2001), Hammond et al.,
"Post-Transcriptional Gene Silencing by Double-Stranded RNA,"
Nature Rev. Gen. 2:110-119 (Abstract) (2001); Hamilton et al., "A
Species of Small Antisense RNA in Posttranscriptional Gene
Silencing in Plants," Science 286:950-952 (Abstract) (1999);
Hammond et al., "An RNA-Directed Nuclease Mediates
Post-Transcriptional Gene Silencing in Drosophila Cells," Nature
404:293-298 (2000); Hutvagner et al., "RNAi: Nature Abhors a
Double-Strand," Curr. Opin. Genetics & Development 12:225-232
(2002), which are hereby incorporated by reference in their
entirety).
[0092] In iRNA, the introduction of double stranded RNA (dsRNA)
into animal or plant cells leads to the destruction of the
endogenous, homologous mRNA, phenocopying a null mutant for that
specific gene. In siRNA, the dsRNA is processed to short
interfering molecules of 21-, 22- or 23-nucleotide RNAs (siRNA),
which are also called "guide RAs," (Hammond et al.,
"Post-Transcriptional Gene Silencing by Double-Stranded RNA,"
Nature Rev. Gen. 2:110-119 (Abstract) (2001); Sharp, "RNA
Interference-2001," Genes Dev. 15:485-490 (2001); Hutvagner et al.,
"RNAi: Nature Abhors a Double-Strand," Curr. Opin. Genetics &
Development 12:225-232 (2002), which are hereby incorporated by
reference in their entirety) in vivo by the Dicer enzyme, a member
of the RNAse III-family of dsRNA-specific ribonucleases (Hutvagner
et al., "RNAi: Nature Abhors a Double-Strand," Curr. Opin. Genetics
& Development 12:225-232 (2002); Bernstein et al., "Role for a
Bidentate Ribonuclease in the Initiation Step of RNA Interference,"
Nature 409:363-366 (2001); Tuschl, "RNA Interference and Small
Interfering RNAs," Chembiochem 2:239-245 (2001); Zamore et al.,
"RNAi: Double Stranded RNA Directs the ATP-Dependent Cleavage of
mRNA at 21 to 23 Nucleotide Intervals," Cell 101:25-3 (2000); U.S.
Pat. No. 6,737,512 to Wu et al., which are hereby incorporated by
reference in their entirety). Successive cleavage events degrade
the RNA to 19-21 bp duplexes, each with 2-nucleotide 3' overhangs
(Hutvagner et al., "RNAi: Nature Abhors a Double-Strand," Curr.
Opin. Genetics & Development 12:225-232 (2002); Bernstein et
al., "Role for a Bidentate Ribonuclease in the Initiation Step of
RNA Interference," Nature 409:363-366 (2001), which are hereby
incorporated by reference in their entirety). The siRNAs are
incorporated into an effector known as the RNA-induced silencing
complex (RISC), which targets the homologous endogenous transcript
by base pairing interactions and cleaves the mRNA approximately 12
nucleotides from the 3' terminus of the siRNA (Hammond et al.,
"Post-Transcriptional Gene Silencing by Double-Stranded RNA,"
Nature Rev. Gen. 2:110-119 (Abstract) (2001); Sharp, "RNA
Interference-2001," Genes Dev. 15:485-490 (2001); Hutvagner et al.,
"RNAi: Nature Abhors a Double-Strand," Curr. Opin. Genetics &
Development 12:225-232 (2002); Nykanen et al., "ATP Requirements
and Small Interfering RNA Structure in the RNA Interference
Pathway," Cell 107:309-321 (2001), which are hereby incorporated by
reference in their entirety).
[0093] There are several methods for preparing siRNA, including
chemical synthesis, in vitro transcription, siRNA expression
vectors, and PCR expression cassettes. In one embodiment, dsRNA for
the nucleic acid molecule used in the present invention can be
generated by transcription in vivo. This involves modifying the
nucleic acid molecule for the production of dsRNA, inserting the
modified nucleic acid molecule into a suitable expression vector
having the appropriate 5' and 3' regulatory nucleotide sequences
operably linked for transcription and translation, as described
supra, and introducing the expression vector having the modified
nucleic acid molecule into a suitable host or subject. Using siRNA
for gene silencing is a rapidly evolving tool in molecular biology,
and guidelines are available in the literature for designing highly
effective siRNA targets and making antisense nucleic acid
constructs for inhibiting endogenous protein (U.S. Pat. No.
6,737,512 to Wu et al.; Brown et al., "RNA Interference in
Mammalian Cell Culture: Design, Execution, and Analysis of the
siRNA Effect," Ambion TechNotes 9(1):3-5(2002); Sui et al., "A DNA
Vector-Based RNAi Technology to Suppress Gene Expression in
Mammalian Cells," Proc. Nat'l. Acad. Sci. USA 99(8):5515-5520
(2002); Yu et al., "RNA Interference by Expression of
Short-Interfering RNAs and Hairpin RNAs in Mammalian Cells," Proc.
Nat'l. Acad. Sci. U.S.A. 99(9):6047-6052 (2002); Paul et al.,
"Effective Expression of Small Interfering RNA in Human Cells,"
Nature Biotechnology 20:505-508 (2002); Brummelkamp et al., "A
System for Stable Expression of Short Interfering RNAs in Mammalian
Cells," Science 296:550-553 (2002), which are hereby incorporated
by reference in their entirety). There are also commercially
available sources for custom-made siRNAs.
[0094] As noted supra, interference of StBEL gene expression is
also achieved in the present invention by the generation of
double-stranded RNA ("dsRNA") through the use of inverted-repeats,
segments of gene-specific sequences oriented in both sense and
antisense orientations. In one embodiment, sequences in the sense
and antisense orientations are linked by a third segment, and
inserted into a suitable expression vector having the appropriate
5' and 3' regulatory nucleotide sequences operably linked for
transcription. The expression vector having the modified nucleic
acid molecule is then inserted into a suitable host cell or
subject. In the present invention, the third segment linking the
two segments of sense and antisense orientation may be any
nucleotide sequence such as a fragment of the .beta.-glucuronidase
("GUS") gene. In another embodiment, a functional (splicing) intron
of the StBEL gene may be used for the third (linking) segment or,
in yet another embodiment of the present invention, other
nucleotide sequences without complementary components in the StBEL
gene may be used to link the two segments of sense and antisense
orientation (Chuang et al., "Specific and Heritable Genetic
Interference by Double-Stranded RNA in Arabidopsis thaliana," Proc.
Nat'l. Academy of Sciences USA 97(9):4985-4990 (2000); Smith et
al., "Total Silencing by Intron-Spliced Hairpin RNAs," Nature
407:319-320 (2000); Waterhouse et al., "Exploring Plant Genomes by
RNA-Induced Gene Silencing," Nature Review: Genetics 4:29-38
(2003); Wesley et al., "Construct Design for Efficient, Effective
and High-Throughput Gene Silencing in Plants," Plant Journal
27(6):581-590 (2001), which are hereby incorporated by reference in
their entirety). In any of the embodiments with inverted repeats of
the StBEL gene, the sense and antisense segments may be oriented
either head-to-head or tail-to-tail in the construct.
[0095] In another embodiment, silencing or reducing expression of
an StBEL using a nucleic acid construct of the present invention
involves using hairpin RNA ("hpRNA"), which may also be
characterized as dsRNA. This involves RNA hybridizing with itself
to form a hairpin structure that comprises a single-stranded loop
region and a base-paired stem. Though a linker may be used between
the inverted repeat segments of sense and antisense sequences to
generate hairpin or double-stranded RNA, the use of intron-free
hpRNA can also be used to achieve silencing of StBEL gene
expression.
[0096] Alternatively, in another embodiment, a plant may be
transformed with constructs encoding both sense and antisense
orientation molecules having separate promoters and no third
segment linking the sense and antisense sequences (Chuang et al.,
"Specific and Heritable Genetic Interference by Double-Stranded RNA
in Arabidopsis thaliana," Proc. Nat'l. Academy of Sciences USA
97(9):4985-4990 (2000); Waterhouse et al., "Exploring Plant Genomes
by RNA-Induced Gene Silencing," Nature Review: Genetics 4:29-38
(2003); Wesley et al., "Construct Design for Efficient, Effective
and High-Throughput Gene Silencing in Plants," Plant Journal
27(6):581-590 (2001), which are hereby incorporated by reference in
their entirety).
[0097] Other means of altering gene expression, including
silencing, are being developed and are also contemplated. For
example, epigenetics is the study of heritable changes in gene
expression or cellular phenotype caused by mechanisms other than
changes in the underlying DNA sequence. Epigenetics refers to
functionally relevant modifications to the genome that do not
involve a change in the nucleotide sequence. Examples of such
changes are DNA methylation and histone deacetylation, both of
which serve to suppress gene expression without altering the
sequence of the silenced genes.
[0098] Enhancing gene expression means increasing the natural or
normal expression of a gene at the level of transcription or
translation. In the present invention, enhancement of StBEL gene
expression may be carried out, according to one embodiment, by a
nucleic acid molecule of the construct containing a functional
StBEL, resulting in increased expression relative to endogenous
StBEL mRNA levels.
[0099] Thus, the constructs of the present invention also include
an operable 3' regulatory region, selected from among those which
are capable of providing correct transcription termination and
polyadenylation of mRNA for expression in the host cell of choice,
operably linked to a DNA molecule which encodes for a protein of
choice. A number of 3' regulatory regions are known in the art.
Virtually any 3' regulatory region known to be operable in the host
cell of choice would suffice for proper expression of the coding
sequence of the nucleic acid designed to enhance expression.
[0100] In one embodiment, the nucleic acid construct of the present
invention has a nucleic acid incorporated into an appropriate
vector to enhance expression, and is positioned in the sense
direction, such that the open reading frame is properly oriented
for the expression of the encoded protein under control of a
promoter of choice. This involves the inclusion of the appropriate
regulatory elements into the DNA-vector construct. These include
non-translated regions of the vector, useful promoters, and 5' and
3' untranslated regions which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, as described infra, may be used.
[0101] A further aspect of the present invention is directed to an
expression vector comprising a nucleic acid construct of the
present invention.
[0102] Another aspect of the present invention is directed to a
host cell transformed with a nucleic acid construct of the present
invention.
[0103] A further aspect of the present invention is directed to a
transgenic plant seed transformed with a nucleic acid construct of
the present invention.
[0104] Another aspect of the present invention is directed to a
transgenic plant transformed with a nucleic acid construct of the
present invention, where the plant has increased tuber yield
compared to a plant not transformed with the nucleic acid
construct.
[0105] A further aspect of the present invention relates to a
transgenic cell of a plant of the present invention.
[0106] Another aspect of the present invention relates to a
transgenic plant seed produced from a plant of the present
invention.
[0107] The nucleotide sequences used in the present invention may
be inserted into any of the many available expression vectors and
cell systems using reagents that are well known in the art.
Suitable vectors include, but are not limited to, the following
viral vectors such as lambda vector system gt11, gt WES.tB, Charon
4, and plasmid vectors such as pG-Cha, p35S-Cha, pBR322, pBR325,
pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,
pKC37, pKC101, SV 40, pBluescript II SK+/- or KS+/-(see "Stratagene
Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif.,
which is hereby incorporated by reference in its entirety), pQE,
pIH821, pGEX, pET series (see Studier et al., "Use of T7 RNA
Polymerase to Direct Expression of Cloned Genes," Gene Expression
Technology vol. 185 (1990), which is hereby incorporated by
reference in its entirety), and any derivatives thereof.
Recombinant molecules can be introduced into cells via
transformation, particularly transduction, conjugation,
mobilization, or electroporation. The DNA sequences are cloned into
the vector using standard cloning procedures in the art, as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y.:Cold Spring Harbor
Press (1989), and Ausubel et al., Current Protocols in Molecular
Biology, New York, N.Y.:John Wiley & Sons (1989), which are
hereby incorporated by reference in their entirety.
[0108] In preparing a nucleic acid construct for expression, the
various nucleic acid sequences may normally be inserted or
substituted into a bacterial plasmid. Any convenient plasmid may be
employed, which will be characterized, for example and without
limitation, by having a bacterial replication system, a marker
which allows for selection in a bacterium, and generally one or
more unique, conveniently located restriction sites. Numerous
plasmids, referred to as transformation vectors, are available for
plant transformation. The selection of a vector will depend on the
preferred transformation technique and target species for
transformation. A variety of vectors are available for stable
transformation using Agrobacterium tumefaciens, a soilborne
bacterium that causes crown gall. Crown gall is characterized by
tumors or galls that develop on the lower stem and main roots of
the infected plant. These tumors are due to the transfer and
incorporation of part of the bacterium plasmid DNA into the plant
chromosomal DNA. This transfer DNA (T-DNA) is expressed along with
the normal genes of the plant cell. The plasmid DNA, pTi, or
Ti-DNA, for "tumor inducing plasmid," contains the vir genes
necessary for movement of the T-DNA into the plant. The T-DNA
carries genes that encode proteins involved in the biosynthesis of
plant regulatory factors, and bacterial nutrients (opines). The
T-DNA is delimited by two 25 bp imperfect direct repeat sequences
called the "border sequences." By removing the oncogene and opine
genes, and replacing them with a gene of interest, it is possible
to transfer foreign DNA into the plant without the formation of
tumors or the multiplication of Agrobacterium tumefaciens (Fraley
et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l
Acad. Sci. 80:4803-4807 (1983), which is hereby incorporated by
reference in its entirety).
[0109] Further improvement of this technique led to the development
of the binary vector system (Bevan, "Binary Agrobacterium Vectors
for Plant Transformation," Nucleic Acids Res. 12:8711-8721 (1984),
which is hereby incorporated by reference in its entirety). In this
system, all the T-DNA sequences (including the borders) are removed
from the pTi, and a second vector containing T-DNA is introduced
into Agrobacterium tumefaciens. This second vector has the
advantage of being replicable in E. coli as well as A. tumefaciens,
and contains a multiclonal site that facilitates the cloning of a
transgene. An example of a commonly-used vector is pBin19 (Frisch
et al., "Complete Sequence of the Binary Vector Bin19," Plant Mol.
Biol. 27:405-409 (1995), which is hereby incorporated by reference
in its entirety). Any appropriate vectors now known or later
described for genetic transformation are suitable for use with the
present invention.
[0110] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby
incorporated by reference in its entirety, describes the production
of expression systems in the form of recombinant plasmids using
restriction enzyme cleavage and ligation with DNA ligase. These
recombinant plasmids are then introduced by means of transformation
and replicated in unicellular cultures including prokaryotic
organisms and eukaryotic cells grown in tissue culture.
[0111] Certain "control elements" or "regulatory sequences" are
also incorporated into the vector-construct. These include
non-translated regions of the vector, promoters, and 5' and 3'
untranslated regions which interact with host cellular proteins to
carry out transcription and translation. Such elements may vary in
their strength and specificity. Depending on the vector system and
host utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. Tissue-specific and organ-specific promoters can also be
used. Native promoters can also be used.
[0112] A constitutive promoter is a promoter that directs
expression of a gene throughout the development and life of an
organism. Examples of some constitutive promoters that are widely
used for inducing expression of transgenes include the nopaline
synthase (NOS) gene promoter, from Agrobacterium tumefaciens (U.S.
Pat. No. 5,034,322 to Rogers et al., which is hereby incorporated
by reference in its entirety), the cauliflower mosaic virus (CaMV)
35S and 19S promoters (U.S. Pat. No. 5,352,605 to Fraley et al.,
which is hereby incorporated by reference in its entirety), those
derived from any of the several actin genes, which are known to be
expressed in most cells types (U.S. Pat. No. 6,002,068 to Privalle
et al., which is hereby incorporated by reference in its entirety),
and the ubiquitin promoter, which is a gene product known to
accumulate in many cell types.
[0113] An inducible promoter is a promoter that is capable of
directly or indirectly activating transcription of one or more DNA
sequences or genes in response to an inducer. In the absence of an
inducer, the DNA sequences or genes will not be transcribed. The
inducer can be a chemical agent, such as a metabolite, growth
regulator, herbicide, or phenolic compound, or a physiological
stress directly imposed upon the plant such as cold, heat, salt,
toxins, or through the action of a pathogen or disease agent such
as a virus or fungus. A plant cell containing an inducible promoter
may be exposed to an inducer by externally applying the inducer to
the cell or plant such as by spraying, watering, heating, or by
exposure to the operative pathogen. An example of an appropriate
inducible promoter is a glucocorticoid-inducible promoter (Schena
et al., "A Steroid-Inducible Gene Expression System for Plant
Cells," Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is hereby
incorporated by reference in its entirety). Expression of the
transgene-encoded protein is induced in the transformed plants when
the transgenic plants are brought into contact with nanomolar
concentrations of a glucocorticoid, or by contact with
dexamethasone, a glucocorticoid analog (Schena et al., "A
Steroid-Inducible Gene Expression System for Plant Cells," Proc.
Natl. Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., "A
Glucocorticoid-Mediated Transcriptional Induction System in
Transgenic Plants," Plant J. 11:605-612 (1997); McNellis et al.,
"Glucocorticoid-Inducible Expression of a Bacterial Avirulence Gene
in Transgenic Arabidopsis Induces Hypersensitive Cell Death," Plant
J. 14(2):247-57 (1998), which are hereby incorporated by reference
in their entirety). In addition, inducible promoters include
promoters that function in a tissue specific manner to regulate the
gene of interest within selected tissues of the plant. Examples of
such tissue specific or developmentally regulated promoters include
seed, flower, fruit, or root specific promoters as are well known
in the field (U.S. Pat. No. 5,750,385 to Shewmaker et al., which is
hereby incorporated by reference in its entirety).
[0114] A number of tissue- and organ-specific promoters have been
developed for use in genetic engineering of plants (Potenza et al.,
"Targeting Transgene Expression in Research, Agricultural, and
Environmental Applications: Promoters used in Plant
Transformation," In Vitro Cell. Dev. Biol. Plant 40:1-22 (2004),
which is hereby incorporated by reference in its entirety).
Examples of such promoters include those that are floral-specific
(Annadana et al., "Cloning of the Chrysanthemum UEP1 Promoter and
Comparative Expression in Florets and Leaves of Dendranthema
grandiflora," Transgenic Res. 11:437-445(2002), which is hereby
incorporated by reference in its entirety), seed-specific (Kluth et
al., "5' Deletion of a gbss1 Promoter Region Leads to Changes in
Tissue and Developmental Specificities," Plant Mol. Biol.
49:669-682 (2002), which is hereby incorporated by reference in its
entirety), root-specific (Yamamoto et al., "Characterization of
cis-acting Sequences Regulating Root-Specific Gene Expression in
Tobacco," Plant Cell 3:371-382 (1991), which is hereby incorporated
by reference in its entirety), fruit-specific (Fraser et al.,
"Evaluation of Transgenic Tomato Plants Expressing an Additional
Phytoene Synthase in a Fruit-Specific Manner," Proc. Natl. Acad.
Sci. USA 99:1092-1097 (2002), which is hereby incorporated by
reference in its entirety), and tuber/storage organ-specific
(Visser et al., "Expression of a Chimaeric Granule-Bound Starch
Synthase-GUS gene in transgenic Potato Plants," Plant Mol. Biol.
17:691-699 (1991), which is hereby incorporated by reference in its
entirety). Targeted expression of an introduced gene (transgene) is
necessary when expression of the transgene could have detrimental
effects if expressed throughout the plant. On the other hand,
silencing a gene throughout a plant could also have negative
effects. However, this problem could be avoided by localizing the
silencing to a region by a tissue-specific promoter. In certain
embodiments, the DNA promoter sequence is a constitutive,
inducible, developmentally-regulated, organelle-specific,
tissue-specific, cell-specific, seed (or grain)-specific, or
germination-specific promoter.
[0115] The nucleic acid constructs of the present invention may
also include an operable 3' regulatory region, selected from among
those which are capable of providing correct transcription
termination and polyadenylation of mRNA for expression in the host
cell of choice, operably linked to a modified trait nucleic acid
molecule of the present invention. A number of 3' regulatory
regions are known to be operable in plants. Exemplary 3' regulatory
regions include, without limitation, the nopaline synthase ("nos")
3' regulatory region (Fraley et al., "Expression of Bacterial Genes
in Plant Cells," Proc. Nat'l Acad. Sci. USA 80:4803-4807 (1983),
which is hereby incorporated by reference in its entirety) and the
cauliflower mosaic virus ("CaMV") 3' regulatory region (Odell et
al., "Identification of DNA Sequences Required for Activity of the
Cauliflower Mosaic Virus 35S Promoter," Nature 313(6005):810-812
(1985), which is hereby incorporated by reference in its entirety).
Virtually any 3' regulatory region known to be operable in plants
would be suitable for use in conjunction with the present
invention.
[0116] As discussed supra, components of nucleic acid constructs
according to the present invention may be heterologous. A
polynucleotide sequence is "heterologous to" an organism or a
second polynucleotide sequence if it is synthetic or originates
from a foreign species or, if from the same species, is modified
from its original form. For example, a promoter operably linked to
a heterologous coding sequence (or vice versa) refers to a coding
sequence from a species different from that from which the promoter
was derived or, if from the same species, a coding sequence which
is not naturally associated with the promoter (e.g., a genetically
engineered coding sequence or an allele from a different ecotype or
variety).
[0117] The different components described above can be ligated
together to produce the expression systems which contain the
nucleic acid constructs used in the present invention, using well
known molecular cloning techniques as described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring
Harbor, N.Y.:Cold Spring Harbor Press (1989), and Ausubel et al.
Current Protocols in Molecular Biology, New York, N.Y.:John Wiley
& Sons (1989), which are hereby incorporated by reference in
their entirety.
[0118] Once the nucleic acid construct has been prepared, it is
ready to be incorporated into a host cell. Basically, this method
is carried out by transforming a host cell with the nucleic acid
construct under conditions effective to achieve transcription of
the nucleic acid molecule in the host cell. This is achieved with
standard cloning procedures known in the art, such as described by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989), which is hereby incorporated by reference in its entirety.
Suitable host cells are plant cells. Suitable host cells also
include bacterial cells. Methods of transformation may result in
transient or stable expression of the nucleic acid under control of
the promoter. In one embodiment, the nucleic acid construct of the
present invention is stably inserted into the genome of the
recombinant plant cell as a result of the transformation, although
transient expression can serve an important purpose, particularly
when the plant under investigation is slow-growing.
[0119] Plant tissue suitable for transformation includes leaf
tissue, root tissue, meristems, zygotic and somatic embryos,
callus, protoplasts, tassels, pollen, embryos, anthers, and the
like. The means of transformation chosen is that most suited to the
tissue to be transformed.
[0120] Transient expression in plant tissue can be achieved by
particle bombardment (Klein et al., "High-Velocity Microprojectiles
for Delivering Nucleic Acids Into Living Cells," Nature 327:70-73
(1987), which is hereby incorporated by reference in its entirety),
also known as biolistic transformation of the host cell, as
disclosed in U.S. Pat. Nos. 4,945,050; 5,036,006; and 5,100,792,
all to Sanford et al., and in Emerschad et al., "Somatic
Embryogenesis and Plant Development from Immature Zygotic Embryos
of Seedless Grapes (Vitis vinifera)," Plant Cell Reports 14:6-12
(1995), which are hereby incorporated by reference in their
entirety.
[0121] In particle bombardment, tungsten or gold microparticles (1
to 2 .mu.m in diameter) are coated with the DNA of interest and
then bombarded at the tissue using high pressure gas. In this way,
it is possible to deliver foreign DNA into the nucleus and obtain a
temporal expression of the gene under the current conditions of the
tissue. Biologically active particles (e.g., dried bacterial cells
containing the vector and heterologous DNA) can also be propelled
into plant cells. Other variations of particle bombardment, now
known or hereafter developed, can also be used.
[0122] An appropriate method of stably introducing a nucleic acid
construct into plant cells is to infect a plant cell with
Agrobacterium tumefaciens or Agrobacterium rhizogenes previously
transformed with the nucleic acid construct. As described supra,
the Ti (or RI) plasmid of Agrobacterium enables the highly
successful transfer of a foreign nucleic acid molecule into plant
cells. A variation of Agrobacterium transformation uses vacuum
infiltration in which whole plants are used (Senior, "Uses of Plant
Gene Silencing," Biotechnology and Genetic Engineering Reviews
15:79-119 (1998), which is hereby incorporated by reference in its
entirety).
[0123] Yet another method of introduction is fusion of protoplasts
with other entities, either minicells, cells, lysosomes, or other
fusible lipid-surfaced bodies (Fraley et al., "Liposome-mediated
Delivery of Tobacco Mosaic Virus RNA Into Tobacco Protoplasts: A
Sensitive Assay for Monitoring Liposome-protoplast Interactions,"
Proc. Natl. Acad. Sci. U.S.A. 79:1859-63 (1982), which is hereby
incorporated by reference in its entirety). The nucleic acid
molecule may also be introduced into the plant cells by
electroporation (Fromm et al., "Expression of Genes Transferred
into Monocot and Dicot Plant Cells by Electroporation," Proc. Natl.
Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by
reference in its entirety). In this technique, plant protoplasts
are electroporated in the presence of plasmids containing the
expression cassette. Electrical impulses of high field strength
reversibly permeabilize biomembranes allowing the introduction of
the plasmids. Electroporated plant protoplasts reform the cell
wall, divide, and regenerate. Other methods of transformation
include polyethylene-mediated plant transformation,
micro-injection, physical abrasives, and laser beams (Senior, "Uses
of Plant Gene Silencing," Biotechnology and Genetic Engineering
Reviews 15:79-119 (1998), which is hereby incorporated by reference
in its entirety). The precise method of transformation is not
critical to the practice of the present invention. Any method that
results in efficient transformation of the host cell of choice is
appropriate for practicing the present invention.
[0124] Yet a further method for introduction is by use of known
techniques for genome editing or alteration. Such techniques for
targeted genomic insertion involve, for example, inducing a double
stranded DNA break precisely at one or more targeted genetic loci
followed by integration of a chosen transgene or nucleic acid
molecule (or construct) during repair. Such techniques or systems
include, for example, zinc finger nucleases ("ZFNs") (Urnov et al.,
"Genome Editing with Engineered Zinc Finger Nucleases," Nat. Rev.
Genet. 11: 636-646 (2010), which is hereby incorporated by
reference in its entirety), transcription activator-like effector
nucleases ("TALENs") (Joung & Sander, "TALENs: A Widely
Applicable Technology for Targeted Genome Editing," Nat. Rev. Mol.
Cell Biol. 14: 49-55 (2013), which is hereby incorporated by
reference in its entirety), clustered regularly interspaced short
palindromic repeat ("CRISPR")-associated endonucleases (e.g.,
CRISPR/CRISPR-associated ("Cas") 9 systems) (Wiedenheft et al.,
"RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,"
Nat. 482:331-338 (2012); Zhang et al., "Multiplex Genome
Engineering Using CRISPR/Cas Systems," Science 339(6121): 819-23
(2013); and Gaj et al., "ZFN, TALEN, and CRISPR/Cas-based Methods
for Genome Engineering," Cell 31(7):397-405 (2013), each of which
is hereby incorporated by reference in its entirety).
[0125] In certain embodiments, transformation described herein is
carried out by Agrobacterium-mediated transformation, whisker
method transformation, vacuum infiltration, biolistic
transformation, electroporation, micro-injection,
polyethylene-mediated transformation, or laser-beam
transformation.
[0126] After transformation, the transformed plant cells must be
regenerated. Plant regeneration from cultured protoplasts is
described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1,
New York, New York: MacMillan Publishing Co. (1983); Vasil, ed.,
Cell Culture and Somatic Cell Genetics of Plants, Vol. I (1984) and
Vol. III (1986), Orlando: Acad. Press; and Fitch et al., "Somatic
Embryogenesis and Plant Regeneration from Immature Zygotic Embryos
of Papaya (Carica papaya L.)," Plant Cell Rep. 9:320 (1990), which
are hereby incorporated by reference in their entirety.
[0127] Means for regeneration vary from species to species of
plants, but generally a suspension of transformed protoplasts or a
petri plate containing explants is first provided. Callus tissue is
formed and shoots may be induced from callus and subsequently
rooted. Alternatively, embryo formation can be induced in the
callus tissue. These embryos germinate as natural embryos to form
plants. The culture media will generally contain various amino
acids and hormones, such as auxin and cytokinins. Efficient
regeneration will depend on the medium, on the genotype, and on the
history of the culture. If these three variables are controlled,
then regeneration is usually reproducible and repeatable.
[0128] In one embodiment, transformed cells are first identified
using a selection marker simultaneously introduced into the host
cells along with the nucleic acid construct of the present
invention. Suitable selection markers include, without limitation,
markers encoding for antibiotic resistance, such as the neomycin
phosphotransferae II ("nptII") gene which confers kanamycin
resistance (Fraley et al., "Expression of Bacterial Genes in Plant
Cells," Proc. Natl. Acad. Sci. U.S.A. 80:4803-4807 (1983), which is
hereby incorporated by reference in its entirety), and the genes
which confer resistance to gentamycin, G418, hygromycin,
streptomycin, spectinomycin, tetracycline, chloramphenicol, and the
like. Cells or tissues are grown on a selection medium containing
the appropriate antibiotic, whereby generally only those
transformants expressing the antibiotic resistance marker continue
to grow. Other types of markers are also suitable for inclusion in
the expression cassette of the present invention. For example, a
gene encoding for herbicide tolerance, such as tolerance to
sulfonylurea is useful, or the dhfr gene, which confers resistance
to methotrexate (Bourouis et al., "Vectors Containing a Prokaryotic
Dihydrofolate Reductase Gene Transform Drosophila Cells to
Methotrexate-resistance," EMBO J. 2:1099-1104 (1983), which is
hereby incorporated by reference in its entirety). Similarly,
"reporter genes," which encode for enzymes providing for production
of an identifiable compound are suitable. The most widely used
reporter gene for gene fusion experiments has been uidA, a gene
from Escherichia coli that encodes the .beta.-glucuronidase
protein, also known as GUS (Jefferson et al., "GUS Fusions: .beta.
Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in
Higher Plants," EMBO J. 6:3901-3907 (1987), which is hereby
incorporated by reference in its entirety). Similarly, enzymes
providing for production of a compound identifiable by
luminescence, such as luciferase, are useful. The selection marker
employed will depend on the target species; for certain target
species, different antibiotics, herbicide, or biosynthesis
selection markers are preferred.
[0129] Plant cells and tissues selected by means of an inhibitory
agent or other selection marker are then tested for the acquisition
of the transgene (Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, New York:Cold Spring Harbor Press
(1989), which is hereby incorporated by reference in its
entirety).
[0130] After the fusion gene containing a nucleic acid construct is
stably incorporated in transgenic plants, the transgene can be
transferred to other plants by sexual crossing. Any of a number of
standard breeding techniques can be used, depending upon the
species to be crossed. Once transgenic plants of this type are
produced, the plants themselves can be cultivated in accordance
with conventional procedure so that the nucleic acid construct is
present in the resulting plants. Alternatively, transgenic seeds
are recovered from the transgenic plants. These seeds can then be
planted in the soil and cultivated using conventional procedures to
produce transgenic plants.
[0131] Plants of the present invention (i.e., having a nucleic acid
construct of the present invention, as discussed supra, or one or
more mutations, as discussed infra) have increased tuber yield.
[0132] In certain embodiments, the transgenic plant transformed
with the nucleic acid construct or plant grown from transgenic
seed, has increased tuber yield compared to a plant not transformed
with the nucleic acid construct. In some embodiments the yield
increase is by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
125%, 150%, 175%, 200%, 250%, or 300% compared to a plant not
transformed with the nucleic acid construct.
[0133] In some embodiments, the overall shoot fresh weight of the
transgenic plant transformed with the nucleic acid construct or
plant grown from transgenic seed is at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 100% compared to a plant not
transformed with the nucleic acid construct.
[0134] In one embodiment, the transgenic plant comprises an
expression level of StBEL11 less than 95%, 90%, 85%, 80%, 75%, 70%,
65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or
1% compared to a plant not transformed with the nucleic acid
construct.
[0135] In some embodiments, the transgenic plant comprises an
expression level of StBEL29 less than 95%, 90%, 85%, 80%, 75%, 70%,
65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or
1% compared to a plant not transformed with the nucleic acid
construct.
[0136] In some embodiments, the transgenic plant comprises an
expression level of StBEL11 and StBEL29 less than 95%, 90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 5%, or 1% compared to a plant not transformed with the
nucleic acid construct.
[0137] In some embodiments, the transgenic plant comprises an
expression level of StBEL5 greater than 1%, 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 5%, 70%, 75%, 80%, 85%, 90%,
95%, 100%, 125%, 150%, 175%, or 200% compared to a plant not
transformed with the nucleic acid construct.
[0138] In some embodiments, the transgenic plant comprises an
expression level of StBEL11 and/or StBEL29 less than 95%, 90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 5%, or 1% compared to a plant not transformed with the
nucleic acid construct, and an expression level of StBEL5 greater
than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
5%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, or 200%
compared to a plant not transformed with the nucleic acid
construct.
[0139] Expression level, as applied to BEL mRNAs, is defined herein
as the level of transcription of the BEL gene. Expression levels
may be quantified by any method known in the art. In one embodiment
accumulation level of BEL is measured in leaves of a young tissue
culture plant using RT-qPCR. In another embodiment, accumulation
level of BEL is measured in stolons of a plant using RT-qPCR. In
another embodiment, accumulation level of BEL is measured in both
the leaves and stolon.
[0140] A person of ordinary skill in the art will understand that
expression levels may be quantified at different developmental
times or in different tissues or cells depending on the nature of
the promoter driving gene expression (e.g. native, constitutive,
inducible, developmentally-regulated, organelle-specific,
tissue-specific, cell-specific, seed specific, or
germination-specific).
[0141] In one embodiment, StBEL 11 and/or StBEL29 is driven by its
natural promoter, and expression levels are measured first in
leaves of a young tissue culture plant using RT-qPCR and
subsequently in stolons of the plant using RT-qPCR.
[0142] In one embodiment, the transgenic plant is selected from the
group consisting of potato, dahlia, caladium, Jerusalem artichoke
(Helianthus tuberosus), yarn (Dioscorea alta), sweet potato
(Impomoea batatus), cassava (Manihot esculenta), tuberous begonia,
cyclamen, other solanum species (e.g., wild potato), sugar beet
(Beta vulgaris), carrot (Daucus carota), and radish (Raphanus
sativus).
[0143] In one embodiment, the transgenic plant is selected from the
group consisting of Solanum tuberosum spp. andigena and Solanum
tuberosum spp. tuberosum.
[0144] Another aspect of the present invention is directed to a
method of increasing tuber yield in a plant. This method involves
providing a transgenic plant or plant seed comprising a nucleic
acid construct comprising one or more nucleic acid molecules
configured to reduce or silence expression of (i) StBEL11 RNA and
variants thereof, (ii) StBEL29 RNA and variants thereof, or (iii)
both (i) and (ii); and growing the transgenic plant or plant grown
from the transgenic plant seed under conditions effective to
express the one or more nucleic acid molecules in said transgenic
plant or said plant grown from the transgenic plant seed.
[0145] In one embodiment, a transgenic plant is provided.
[0146] In another embodiment, a transgenic seed is provided.
[0147] In a further embodiment, providing comprises transforming a
non-transgenic plant or a non-transgenic plant seed with the
nucleic acid construct to yield the transgenic plant or plant
seed.
[0148] Providing a transgenic plant or plant seed may include
transforming a non-transgenic plant or a non-transgenic plant seed
with the nucleic acid construct to yield said transgenic plant or
plant seed. Suitable methods of transformation are described
supra.
[0149] Tuber yield as used herein can be measured as fresh weight
of tubers per plant or dry weight of tubers per plant.
[0150] The increased tuber yield may be by any amount. For example,
the increased tuber yield may be by about (or by at least about)
1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%,
250%, or 300% compared to a plant not transformed with the nucleic
acid construct.
[0151] Increased tuber yield, as well as any other trait described
herein (e.g., overall shoot fresh weight, etc.) in a plant as
described herein may be determined in comparison to a control
plant. The choice of suitable control plants is a routine part of
an experimental setup and may include corresponding wild type
plants, corresponding plants without the gene of interest (e.g.,
those not transformed with the subject nucleic acid molecule). The
control plant is typically of the same plant species or even of the
same variety as the plant to be assessed. The control plant may
also be a nullizygote of the plant to be assessed. A "control
plant" as used herein refers not only to whole plants, but also to
plant parts, including inflorescence, seeds, and seed parts.
[0152] Yet another aspect of the present invention is directed to a
potato plant comprising one or more mutations in one or both of
StBEL11 and StBEL29, wherein said potato plant has increased tuber
yield compared to the tuber yield of a wild type potato plant.
[0153] In one embodiment, the potato plant further comprising one
or more mutations in StBEL5.
[0154] In one embodiment, the potato plant is non-transgenic.
Accordingly, it is contemplated that mutations leading to increased
tuber yield may be introduced via gene editing techniques or
induced mutation techniques. Alternatively, naturally occurring
mutations may be combined via traditional breeding to produce high
tuber yield plants.
[0155] In one embodiment, the mutated potato plant is obtained by
subjecting at least one cell of a potato plant to a chemical
mutagenizing agent under conditions effective to yield at least one
mutant plant cell containing an inactive or partially inactive
StBEL gene or variant thereof. A suitable chemical mutagenizing
agent can include, for example, ethylmethanesulfonate.
[0156] In another embodiment, the mutated potato plant is obtained
by subjecting at least one cell of a potato plant to a radiation
source under conditions effective to yield at least one mutant
plant cell containing an StBEL gene or variant thereof with altered
expression. Suitable radiation sources can include, for example,
sources that are effective in producing ultraviolet rays, gamma
rays, or fast neutrons.
[0157] In another embodiment, the mutated potato plant is obtained
by inserting an inactivating nucleic acid molecule into the gene
encoding the functional StBEL gene or its promoter under conditions
effective to inactivate the gene. Suitable inactivating nucleic
acid molecules can include, for example, a transposable element.
Examples of such transposable elements include, but are not limited
to, an Activator (Ac) transposon, a Dissociator (Ds) transposon, or
a Mutator (Mu) transposon.
[0158] In yet another embodiment, the mutated potato plant is
obtained by subjecting at least one cell of a potato plant to
Agrobacterium transformation under conditions effective to insert
an Agrobacterium T-DNA sequence into the gene, thereby inactivating
the gene. Suitable Agrobacterium T-DNA sequences can include, for
example, those sequences that are carried on a binary
transformation vector of pAC106, pAC161, pGABI1, pADIS1, pCSA110,
pDAP101, derivatives of pBIN19, or pCAMBIA plasmid series.
[0159] In yet another embodiment, the mutated potato plant is
obtained by subjecting at least one cell of a potato plant to
site-directed mutagenesis of the StBEL gene or its promoter under
conditions effective to yield at least one mutant plant cell
containing an StBEL gene with altered expression. See, e.g., Baker,
"Gene-editing Nucleases," Nature Methods 9(1):23-26 (2012), which
is hereby incorporated by reference in its entirety. The treating
step may also involve subjecting the at least one cell of the
potato plant to site-directed mutagenesis of the StBEL gene under
conditions effective to yield at least one mutant plant cell
containing a variant StBEL gene associated with increased tuber
yield as described herein above. The various plants that can be
used in this method are the same as those described supra with
respect to the transgenic plants and mutant plants.
[0160] In yet another embodiment, the mutated potato plant is
obtained by subjecting at least one cell of a potato plant to gene
editing, as described supra, to yield at least one mutant plant
cell containing a modified (or variant) sequence of the StBEL gene
associated with tuber yield as described herein above. Propagating
the at least one mutant plant cell into a mutant plant results in a
mutant plant having an altered level of StBEL protein or variant
thereof associated with tuber production as described herein above
compared to that of the nonmutant plant and displays an altered
(e.g., increased) tuber yield phenotype relative to a nonmutant
plant at the levels or amounts discussed supra.
[0161] In one embodiment, the potato plant comprises a reduced
expression level of StBEL11 compared to a wild type potato plant at
the levels discussed supra.
[0162] In another embodiment, the expression level of StBEL11 is
measured by quantifying accumulation levels of StBEL11 in leaves of
a young tissue culture plant using RT-qPCR.
[0163] In a further embodiment, the potato plant comprises a
reduced expression level of StBEL29 compared to a wild type potato
plant at the levels discussed supra.
[0164] In one embodiment, the expression level of StBEL29 is
measured by quantifying accumulation levels of StBEL11 in leaves of
a young tissue culture plant using RT-qPCR, as discussed supra.
[0165] In one embodiment, the potato plant comprises a tuber yield
at a level discussed supra.
[0166] In one embodiment, the potato plant comprises an increased
expression level of StBEL5 compared to a wild type potato plant at
the levels discussed supra.
[0167] The present invention is also directed to potato seed from
the potato plants described herein.
EXAMPLES
Example 1--Cloning Strategies and Whole-Plant Transformation
[0168] Partial cDNA sequences of both StBEL11 (GenBank: AF406698,
which is hereby incorporated by reference in its entirety) and
StBEL29 (GenBank: AF406702, which is hereby incorporated by
reference in its entirety) were obtained from NCBI. Putative
upstream sequences were identified using the potato genome database
(http://potato.plantbiology.msu.edu). Genomic DNA from leaves of
wild-type potato (Solanum tuberosum ssp. andigena) was isolated
using DNeasy plant mini kit (QIAGEN). Upstream regulatory sequences
of both StBEL11 and StBEL29 genes were isolated using the Universal
Genome Walker kit (Clontech). Both these sequences were verified
using the online potato genome database. Upstream sequences of 1678
bp for StBEL11 and 2151 bp for StBEL29 were isolated from the
genomic DNA of Solanum tuberosum ssp. andigena through genome
walking. The upstream sequences of both genes were amplified using
gene-specific primers (Table 1) and were verified by sequencing.
Both sequences were fused to the .beta.-glucoronidase (GUS) gene
and cloned into the binary vector pBI121 to generate the
proStBEL11:GUS and proStBEL29:GUS constructs.
[0169] Full-length sequences of StBEL11 (2718 bp) (SEQ ID NO:2) and
StBEL29 (2898 bp) (SEQ ID NO:5) were PCR amplified with
gene-specific primers (Table 1) and were cloned into the binary
vectors pBI121 and pCAMBIA1300 respectively, under the CaMV 35S
promoter to generate the 35S:StBEL11 and 35S:StBEL29 constructs.
These constructs were then transformed into Agrobacterium
tumefaciens strain GV2260. Stably transformed lines were generated
and ten lines of each type were selected for further expression and
phenotypic analyses (FIGS. 1A-1B).
[0170] The GAS:GUS construct created in pBI101.2 was described
previously (Banerjee et al., "Dynamics of a Mobile RNA of Potato
Involved in a Long-Distance Signaling Pathway," Plant Cell
18:3443-57 (2006), which is hereby incorporated by reference in its
entirety).
[0171] For generating the GAS:StBEL11 construct, the full-length
StBEL11 cDNA was cloned into the XmaI/SacI site downstream from the
GAS promoter, cloned previously into pBI101.2 (Banerjee et al.,
"Dynamics of a Mobile RNA of Potato Involved in a Long-Distance
Signaling Pathway," Plant Cell 18:3443-57 (2006), which is hereby
incorporated by reference in its entirety).
[0172] The GAS:StBEL29 construct was generated by cloning the
full-length StBEL29, which was PCR amplified with primers flanking
the 5' XmaI and 3' EcoRV sites, into the XmaI/SacI (blunt-ended)
sites of pBI101.2 with the GAS promoter inserted previously
(Banerjee et al., "Dynamics of a Mobile RNA of Potato Involved in a
Long-Distance Signaling Pathway," Plant Cell 18:3443-57 (2006),
which is hereby incorporated by reference in its entirety).
[0173] All constructs were confirmed via sequencing at the DNA
Facility at Iowa State University.
[0174] These constructs were transformed into Agrobacterium
tumefaciens strain GV2260. Wild-type potato leaves were transformed
and transgenic plants were generated by Agrobacterium-mediated
transformation as reported previously (Banerjee et al., "Efficient
Production of Transgenic Potato (S. tuberosum L. ssp. andigena)
Plants via Agrobacterium Tumefaciens-Mediated Transformation,"
Plant Sci. 170:732-38 (2006), which is hereby incorporated by
reference in its entirety). Transgenic lines for each of the four
constructs were screened for level of transgene expression in at
least ten independent transgenic lines, except in the case of the
GAS:StBEL11 construct, where 6 lines were screened (FIGS. 1A-1B and
2A-2B). The in vitro transgenic potato plants were maintained in a
growth chamber (Percival Scientific Inc.) at 27.degree. C. with a
photoperiod of 16 h light, 8 h dark and a fluence rate of 40
.mu.mol m.sup.-2 s.sup.-1. Soil-grown plants were maintained in a
growth chamber under either a long-day (16 h light at 22.degree.
C., 8 h dark at 18.degree. C.) or short-day (8 h light at
22.degree. C., 16 h dark at 18.degree. C.) photoperiod with a
fluence rate of 400 .mu.mol m.sup.-2 s.sup.-1.
TABLE-US-00009 TABLE 1 Primers Cloning full-length sequences of
StBEL11 and StBEL29 under 35S SEQ ID constitutive promoter NO:
BEL11F CATCTAGAGTAGGGGGGGAGGCACC 9 BEL11R
GAGAGCTCGAAGCACAAAATTTACAATATAC 10 BEL29F
ACATCTAGATTAGCTCTCATCACTTCACA 11 BEL29R AGAGGTACCTACCACCCAAAATACTAC
12 SEQ ID Primers for generating StBEL11 and StBEL29 suppression
lines NO: StBEL11AS FP GAGCTCGAAATTTATGGCTATGTACTATC 13 StBEL11AS
RP TCTAGAGTGGAAGACGGTATATGTGAT 14 StBEL29AS FP
GAGCTCGTGTTATTTGTTTATTGTGGAGA 15 StBEL29AS RP
TCTAGAGTCTGCTCCAACTCCGTCTA 16 SEQ ID StBEL11 and StBEL29 promoter
cloning NO: PBEL11F TATAAGCTTAACTAACTAACTAACTGTCCC 17 PBEL11R
GAGTCTAGAACTCCACAACACATAAAGGG 18 PBEL29F
CACAAGCTTTGAGAAGAAAACCAAAGAAAC 19 PBEL29R
AACGGATCCAGATGTGGATGTGTGAATGTG 20 SEQ ID Cloning of StBEL11 and
StBEL29 under GAS promoter NO: GASBEL11f
TATATTATATCCCGGGTTTAAGAAAATCTCTCACTTTC 21 TCT GASBEL11r
TATATTATATGAGCTCGTTTACATATATGCAAATTGA 22 AACA GASBEL29f
TATATTATATCCCGGGTTCTTTCTTTCTTTCTCCTCTCT 23 CT GASBEL29r
TATATTATATGATATCGGCTAAAATGGATGGAGTATT 24 ATTT SEQ ID RT-qPCR of
StBEL11, StBEL29 and GAPDH NO: 11F AGGACACTAGCAAAACTTTAGG 25 11R
CTTTGAGGC TTCCATGCATTG 26 29F CATTTGCCTCAACACAACCC 27 29R
TGATGCTTTCGATCTCTGGTG 28 GAPDH-F GAAGGACTGGAGAGGTGGA 29 GAPDH-R
GACAAC AGAAACATCAGCAGT 30 SEQ ID Transgenic-specific primers used
for heterografts and screening NO: NT-142 GCGGGACTCTAATCATAAAAAC 31
GUS-GSP1 TGGAAACGGCAGAGAAGGTAC 32 29-GSP1 ATTAGTGACGGACAACTTCTGTC
33 11-GSP1 GTAAATCAGCTTGAAATTACATCATG 34 SEQ ID Primers for
confirming StBEL11 and StBEL29 suppression lines NO: StBEL11AS RP
TCTAGAGTGGAAGACGGTATATGTGAT 35 StBEL29AS RP CAGAAAATCCAACATATCCAG
36 NOST RPscr GCAACAGGATTCAATCTTAAG 37 KanR FP GGATTGCACGCAGGTTCT
38 KanR RP CGTCAAGAAGGCGATAGAA 39 SEQ ID Transgene-specific primers
used for RT-qPCR in RNA movement assays NO: B11MqRT (GSP)
CTATATATGCAAACTATAGTATGTTG 40 B29MqRT (GSP)
CTTCTAGAAGATATATATATGGTTGAG 41 NTR (vector specific)
GCAACAGGATTCAATCTTAAGAAACT 42 SEQ ID RT-qPCR (Analysis of target
genes) NO: StACTqRTf GGAAAAGCTTGCCTATGTGG 43 StACTqRTr
CTGCTCCTGGCAGTTTCAA 44 StPIN2qRTf TCATCTAAAGGGCCAACACC 45
StPIN2qRTr GTTGTATAGCTCCCCGCTCA 46 StGA2ox1qRTf
TTCTCTACAATGAGTTCACATGGTC 47 StGA2ox1qRTr GGGACAACCTATTATCACCAAGC
48 StIAA3qRTf CTGATCTTCGATCAATTTCATGG 49 StIAA3qRTr
GACCTATTGCTGCCTTGTGCTA 50
Example 2--RNA Suppression Analysis
[0175] To generate transgenic suppression lines for StBEL11 and
StBEL29, non-conserved antisense sequences were used to design
constructs. The antisense fragments (401 and 799 bp of StBEL11 and
StBEL29 cDNAs, corresponding to SEQ ID NO:3 and SEQ ID NO:6,
respectively) contained coding sequence and a small portion of the
5' UTR. These were amplified and cloned in the antisense direction
into the binary vector pCB201 driven by the CaMV 35S promoter
(Xiang et al., "A Mini Binary Vector Series for Plant
Transformation," Plant Mol. Biol. 40:711-17 (1999), which is hereby
incorporated by reference in its entirety).
[0176] Potato (Solanum tuberosum ssp. andigena) leaf transformation
was performed with these two constructs as described above. Stable
transformants were confirmed using PCR of genomic DNA of in vitro
plantlets using gene-specific primers (Table 1). At least nine
independent transgenic lines (ten plants per transgenic line) for
each construct were screened for a reduction in StBEL11 and StBEL29
transcript levels in both the leaves of one-month old soilgrown
long-day plants and stolons from select transgenic lines grown
under short-days for 21 days (FIGS. 3A-D). Soil-grown plants (ten
plants per transgenic line) were maintained in a growth chamber
under a short-day (8 h light at 22.degree. C., 16 h dark at
18.degree. C.) photoperiod with a fluence rate of 400 .mu.mol
m.sup.-2 s.sup.-1. Two lines for each construct, designated 11-1,
11-2, 29-1, 29-2, were selected for further analyses.
[0177] Expression of the tuber marker gene StSP6A was quantified
from stolons of the two selected lines per construct. For RT-qPCR
analysis, stolons were pooled from three independent plants off ten
plants per transgenic line forming three biological replicates per
line. Total RNA was isolated from ground tissues using RNAiso Plus
(Takara-Clontech) and two micrograms of RNA (DNase treated with RQ1
RNase-Free DNase; Cat.# M6101; Promega) were reverse-transcribed
using oligo(dT) primer and SuperScript-III reverse transcriptase
(Invitrogen). qPCR was performed on a CFX96 Real-Time System
(BIO-RAD) using gene-specific primers (Table 1). The reactions were
carried out using KAPA SYBR.RTM. green master mix (KAPA Biosystems)
and incubated at 95.degree. C. for 2 min, followed by 40 cycles of
95.degree. C. for 15 s and 60.degree. C. for 30 s. GAPDH was used
for normalization for all the reactions (Table 1). PCR specificity
was checked by melting curve analysis, and data were analyzed using
the 2.sup.-.DELTA..DELTA.ct method (Livak & Schmittgen,
"Analysis of Relative Gene Expression Data Using Real-Time
Quantitative PCR and the 2(-delta delta C(T)) Method," Methods
25:402-08 (2001), which is hereby incorporated by reference in its
entirety). Shoot growth and tuber yield from these antisense lines
were also measured. Statistical analysis was carried out with the
Student's t test using GraphPad Prism (6.07 version).
Example 3--Histochemical and Fluorometric Assay
[0178] For qualitative GUS assays, samples were incubated in GUS
staining buffer containing (1.0 M NaPO.sub.4 pH 7, 0.25 M EDTA pH
8, 0.05 mM potassium ferricyanide, 0.05 mM potassium ferrocyanide
and 1.0 mM X-gluc) for 16 h at 37.degree. C. Samples were then
washed with 100% ethanol. For histology, stained petioles and stems
were cut into 0.5 cm long pieces and imbedded into 4% agarose
blocks. Sections were obtained using a Leica vibratome VT1200. All
samples were visualized using a Leica microscope (S8AP0). For
fluorometric analysis, frozen tissue samples were ground in GUS
extraction buffer (50 mM NaPO.sub.4 pH 7, 10 mM EDTA pH 8.0, 10 mM
.beta.-mercaptoethanol, 0.1% Triton.TM. X-100 0.1% SDS) as
described by Jefferson et al., "Assaying Chimeric Genes in Plants:
The GUS Gene Fusion System," Plant Mol. Biol. Rep. 5:387-405
(1987), which is hereby incorporated by reference in its entirety.
Samples were centrifuged at 17,000 rpm for 5 min. This was followed
by protein quantification using the Bradford assay (Bradford et
al., "A Rapid and Sensitive Method for the Quantitation of
Microgram Quantities of Protein Utilizing the Principle of
Protein-Dye Binding," Anal. Biohem. 72:248-254 (1976), which is
hereby incorporated by reference in its entirety. Approximately 10
.mu.s of the total protein was aliquoted with 5 .mu.l of GUS assay
buffer (50 mM MUG) and samples were incubated at 37.degree. C. for
16 h. The reaction was stopped by adding stop buffer (0.2 M
Na.sub.2CO.sub.3). GUS activity was monitored at emission
wavelength 365 nm and excitation wavelength 455 nm using a
Varioskan flash plate reader (Thermo Scientific).
Example 4--Heterografts
[0179] Simple splice micrografts under sterile conditions were made
using material from 4-week old GAS:StBEL11, GAS:StBEL29, or GAS:GUS
transgenic lines for scion material (shoots with 3-5 leaves) and
4-week old wildtype andigena for stocks (rooted stems approximately
1.5 cm in length). The micrografts were grown in vitro for 2 weeks
before transfer to soil. In soil, the heterografts were then grown
for 3 weeks under long-day conditions (16 h of light, 8 h of dark,
25.degree. C.), followed by 2 weeks under short-days (8 h of light,
16 h of dark, 25.degree. C.) before sample harvest, RNA extraction,
and a single round of gel-based RT-PCR using transgenic
gene-specific primers (Table 1).
Example 5--RT-qPCR Analysis
[0180] Eight-week old soil-grown wild-type andigena potato plants
were grown under either SD or LD conditions for 15 days in a growth
chamber (Percival Scientific). Leaf, petiole, stem, root, and
stolon samples were then harvested in liquid nitrogen, ground and
stored at -80.degree. C. RNA was isolated from frozen samples using
the RNeasy plant mini kit (QIAGEN). To avoid genomic DNA
contamination, total RNA was treated with RNase-free DNase Set
(QIAGEN) and quantified.
[0181] Gene-specific cDNAs for StBEL11, StBEL29, and GAPDH were
prepared with 2.0 .mu.g of total RNA using MMLV reverse
transcriptase (Promega). RTqPCR was performed in a 10 .mu.l
reaction volume with primer concentrations of 0.3 .mu.M and 1 .mu.l
of cDNA and KAPA SYBR.RTM. mastermix. The reaction mix was
incubated at 95.degree. C. for 3 min, followed by 40 cycles at
95.degree. C. for 10 s, 55.degree. C. for 20 s, and 60.degree. C.
for 20 s.
[0182] PCR specificity was confirmed by melting curve analysis and
data were analyzed using 2.sup..DELTA..DELTA.ct method (Livak &
Schmittgen, "Analysis of Relative Gene Expression Data Using
Real-Time Quantitative PCR and the 2(-delta delta C(T)) Method,"
Methods 25:402-08 (2001), which is hereby incorporated by reference
in its entirety). GAPDH or StActin8 were used as internal controls.
Gene specific primers for StBEL11, StBEL29, and GAPDH genes were
used (Table 1).
[0183] Polysomal RNA extraction was performed as previously
described (Mignery et al., "Isolation and Sequence Analysis of
cDNAs for the Major Potato Tuber Protein, Patatin," Nucleic Acids
Res. 12:7987-8000 (1984), which is hereby incorporated by reference
in its entirety). Gene-specific primers (Table 1) with RT-qPCR were
also used for the target gene assays. For all RT-qPCR analyses, the
average of two or three technical replicates was first taken into
consideration, followed by the statistical analyses of two or three
biological replicates.
Example 6--Phenotypes of StBEL11 and StBEL29 in Both Overexpression
and Suppression Lines
[0184] Because of the close sequence similarity among StBEL5,
StBEL11, and StBEL29 (Sharma et al., "The BEL1-Like Family of
Transcription Factors in Potato," J. Expt. Bot. 65:709-23 (2014),
which is hereby incorporated by reference in its entirety), the
possibility that StBEL11 and StBEL29 may be co-functional with
StBEL5 in tuber formation was considered. To better understand the
function of StBEL11 and StBEL29, approximately ten transgenic
CaMV-35S over-expression (OE) lines of S. tuberosum ssp. andigena
were generated for both StBEL types, screened and evaluated (FIGS.
1A-1B). Two independent lines that exhibited substantial levels of
transcripts of StBEL11 and StBEL29 were used in evaluating effects
on phenotypes of soil-grown plants (FIGS. 4A-4B). Overall shoot
fresh weight was not affected in any of the OE lines (FIG. 4A), but
a significant reduction in tuber yields was observed in all four
transgenic lines (FIG. 4B).
[0185] To further validate the phenotype produced by the expression
of StBEL11 and StBEL29, antisense constructs specific for each
respective gene were transformed into S. tuberosum ssp. andigena.
Independent lines were double screened in both leaves and stolons
for suppression (FIGS. 3A-3D). Suppression levels in stolons
relative to WT (wild-type) ranged from 0.3 to no suppression (FIGS.
3A-3D). From this group, two lines per construct were selected,
designated as 29-1, 29-2 for StBEL29 and 11-1, 11-2 for StBEL11,
and were evaluated for shoot growth and tuber yields (FIGS. 5A-5C).
Whereas, there was very little difference in shoot growth between
WT and the antisense lines (FIG. 5A), tuber yields increased
substantially under the short-day conditions (FIG. 5B). Among these
four lines, increases in tuber yields ranged from two- to
three-fold.
[0186] Overall tuber numbers per plant increased in these
transgenic lines but the morphology of the tubers from the
antisense lines appeared to be comparable to WT (FIGS. 6A-6C). To
verify that this yield effect was mediated through the tuberization
signaling pathway, expression of the tuber signal gene, StSP6A, was
quantified in stolons of all four lines (FIG. 5C). Mean transcript
levels of StSP6A increased by as much as five-fold relative to the
control, and were closely correlated to overall tuber yield
increase (FIGS. 5B, 5C). These results suggest that similar to
StBEL5 (Sharma et al., "Targets of the StBEL5 Transcription Factor
Include the FT Ortholog StSP6A," Plant Physiol. 170:310-24 (2016),
which is hereby incorporated by reference in its entirety), StSP6A
is targeted by StBEL11 and StBEL29 to control its transcriptional
activity and regulate the tuberization pathway.
Example 7--Transcriptional Activity of StBEL11 and StBEL29
[0187] To assess expression patterns in whole plants, upstream
sequences of the StBEL11 and StBEL29 genes were isolated and fused
to .beta.-glucoronidase gene (GUS) and cloned into the binary
vector pBI121 to generate proStBEL11:GUS and proStBEL29:GUS
constructs (FIG. 7A). These were transformed into potato, S.
tuberosum ssp. andigena, and ten lines for each construct were
screened through histochemical GUS expression assays. Robust GUS
activity was observed in primary leaf veins, petioles, and stems of
both proStBEL11:GUS (FIGS. 7B, 7D, 7E, arrows) and proStBEL29:GUS
(FIGS. 7F-7H, arrows) lines. GUS expression was not observed in
roots of any of the in vitro grown transgenic lines tested (FIGS.
7B, 7H), and only faint GUS staining was observed in stolons from
21 day SD-induced plants (FIGS. 7C, 7I). Previous studies had shown
the presence of StBEL11 and StBEL29 transcripts in phloem cells of
potato (Yu et al., "Tissue Integrity and RNA Quality of Laser
Microdissected Phloem of Potato," Planta 226:797-803 (2007), which
is hereby incorporated by reference in its entirety). RNA-seq data
from phloem-associated cells of petioles and stems (Lin et al.,
"Transcriptional Analysis of Phloem-Associated Cells of Potato,"
BMC Genom. 16:665 (2015), which is hereby incorporated by reference
in its entirety) revealed substantial amounts of RNA from several
StBEL genes accumulating in these cells (Table 2). The greatest
levels were observed for the mobile RNA, StBEL5, and for StBEL29
(Table 2).
TABLE-US-00010 TABLE 2 Accumulation of Known Mobile mRNAs (*) and
Selected StBELs in Petiole and Stem Phloem-Associated Cells Petiole
Stem Annotation Gene ID phloem phloem Function Citation StGAI*
PGSC0003DMG400015692 531 422 Leaf .sup.aHaywood morphology POTH1*
PGSC0003DMG400013493 267 24 Vegetative .sup.bMahajan growth StBEL5*
PGSC0003DMG400005930 2089 1234 Tuber/root .sup.cBanerjee growth
StBEL11* PGSC0003DMG400019635 92 85 Tuber growth This report
StBEL29* PGSC0003DMG400021323 1282 2591 Tuber growth This report
StBEL33 PGSC0003DMG400024267 464 812 Unknown .sup.eSharma StBEL34
PGSC0003DMG400008057 199 66 Unknown .sup.eSharma StBEL35
PGSC0003DMG400019142 301 453 Unknown .sup.eSharma *indicates known
mobile mRNAs. .sup.aHaywood et al., "Phloem Long-Distance
Trafficking of GIBBERELLIC ACID-INSENSITIVE RNA Regulates Leaf
Development," Plant J. 42: 49-68 (2005), which is hereby
incorporated by reference in its entirety. .sup.bMahajan et al.,
"The mRNA of a Knotted1-Like Transcription Factor of Potato is
Phloem Mobile," Plant Mol. Biol. 79: 595-608 (2012), which is
hereby incorporated by reference in its entirety. .sup.cBanerjee et
al., "Dynamics of a Mobile RNA of Potato Involved in a
Long-Distance Signaling Pathway," Plant Cell 18: 3443-57 (2006),
which is hereby incorporated by reference in its entirety.
.sup.dSharma et al., "The BEL1-Like Family of Transcription Factors
in Potato," J. Expt. Bot. 65: 709-23 (2014), which is hereby
incorporated by reference in its entirety.
[0188] The values for petiole and stem phloem are the means of the
number of reads for three replicates of RNA-seq data from Lin et
al., "Transcriptional Analysis of Phloem-Associated Cells of
Potato," BMC Genom. 16:665 (2015), which is hereby incorporated by
reference in its entirety. After sequencing, reads were processed
and aligned to the potato genome. The number of concordant unique
reads in each library was counted with HTseq, and the three
libraries were normalized with the 0.75 quantile to eliminate the
differences caused by the sample scale and sequencing depth.
[0189] To assess the cellular location of promoter activity for
StBEL11 and StBEL29 in vascular cells of petioles and stems,
histochemical analysis was performed on samples taken from
soil-grown proStBEL11:GUS and proStBEL29:GUS transgenic lines grown
under short-day conditions. GUS activity was visually assessed in
transverse sections of both tissue types (FIGS. 8A-8D). Strong GUS
activity was observed for both promoter lines in external phloem
cells and in xylem cell walls in petioles for both StBEL11 and
StBEL29 promoter transgenic lines (FIGS. 8A, 8B, arrows). GUS
signal was also specifically observed in xylem cell walls and in
double layers of the epidermis in stems of StBEL11 (FIG. 8C,
arrows) and in phloem and xylem cell walls of stems for StBEL29
(FIG. 8D, arrows).
Example 8--Effect of Photoperiod on StBEL11 and StBEL29 mRNA
Accumulation Patterns
[0190] Phylogenetic analysis of the thirteen BEL TFs identified
from potato revealed that StBEL11 and StBEL29 exhibited a very
close amino acid sequence match with StBEL5 (Chen et al.,
"Interacting Transcription Factors From the Three Amino Acid Loop
Extension Superclass Regulate Tuber Formation," Plant Physiol.
132:1391-1404 (2003) and Sharma et al., "The BEL1-Like Family of
Transcription Factors in Potato," J. Expt. Bot. 65:709-23 (2014),
which are hereby incorporated by reference in their entirety).
StBEL5 mRNA accumulation and mobility were enhanced by short-days
in a transport-mediated process (Banerjee et al., "Dynamics of a
Mobile RNA of Potato Involved in a Long-Distance Signaling
Pathway," Plant Cell 18:3443-57 (2006) and Cho et al.,
"Polypyrimidine Tract-Binding Proteins of Potato Mediate
Tuberization Through an Interaction With StBEL5 RNA," J. Expt. Bot.
66:6835-47 (2015), which are hereby incorporated by reference in
their entirety).
[0191] Because photoperiod is an important cue for regulating the
onset of tuberization, and to determine if photoperiod had any
effect on the steady-state levels of StBEL11 and StBEL29 mRNAs,
total and polysomal RNA levels for both were measured in leaves,
petioles, stem, roots, and stolons from LD and SD andigena plants
(FIGS. 9A-9D). Transcript accumulation of both StBEL11 and StBEL29
occurred throughout the plant under both LD and SD photoperiods,
but organ-specific differential accumulation of mRNAs was observed
when plants were grown under SD conditions (FIGS. 9A, 9B).
[0192] Significant differences in mRNA levels were observed for
petioles, stems, and stolons for both StBEL types. In the tested
samples, RNA levels were less in leaves in comparison to petiole
and stems. RNA levels of both StBEL11 and StBEL29 were similar in
roots under both photoperiodic conditions. Among all the organs
evaluated, stolons exhibited the greatest RNA accumulation under SD
conditions for both StBEL11 and StBEL29 RNAs. Similar patterns of
accumulation were also observed for polysomal RNA fractions for
both of the StBEL1-types (FIGS. 9C, 9D).
[0193] In theory, polysomal RNA is a measure of mRNAs that are
being actively translated in these organs. This analysis indicates
that transcript accumulation patterns varied among the organs
tested, but that levels in petioles, stems, and stolons were
significantly affected by photoperiod. Levels of total RNA in
stolons from SD plants increased 7.6- and 10-fold for StBEL11 and
StBEL29, respectively. Similar enhancement levels were observed in
the polysomal fractions (FIGS. 9C, 9D). To better understand, the
effect of photoperiod on StBEL11 and StBEL29 patterns of transcript
accumulation, promoter activity of both types were quantified under
both LD and SD conditions in several organs (FIGS. 10A, 10B). Both
StBEL1-types responded to photoperiod in a similar fashion.
Promoter activity was induced by SD in leaves, petioles, and
stolons. Activity in roots was slightly enhanced by LD conditions.
No difference in promoter activity was observed in stems (FIGS.
10A, 10B).
Example 9--StBEL11 and StBEL29 RNAs are Phloem Mobile
[0194] Because StBEL11 and StBEL29 transcription occurs in vascular
cells of both stems and petioles and in light of the liberal
mobility of StBEL5 RNA, heterografts were implemented to assess the
capacity of these RNAs for long-distance transport. Heterografts
were composed of transgenic scions with a GAS (galactinol synthase)
promoter driving full-length StBEL11 or -29 expression and
wild-type (WT) stocks (FIGS. 11A-11C). The GAS promoter is
specifically expressed in minor veins of the leaf (Ayre et al.,
"Functional and Phylogenetic Analyses of A Conserved Regulatory
Program in the Phloem of Minor Veins," Plant Physiol. 133:1229-39
(2003), which is hereby incorporated by reference in its entirety).
After grafts formed in vitro and several weeks of growth in soil,
RTPCR with transgene-specific primers was performed in secondary
roots and stolons of the WT andigena stock. In all four replicates
for both StBEL11 and StBEL29, transgenic RNA was detected in
stolons tips and secondary roots of the WT stock (FIGS. 11A, 11B).
No GUS transcripts were detected in WT stocks of the GAS:GUS/WT
heterografts and no transgenic RNAs were detected in WT/WT grafts
(FIGS. 11C, 11D). These results clearly demonstrate that the
leaf-derived transcripts of both StBEL11 and StBEL29 move down into
secondary roots and stolon tips through the sieve element system
when expressed from the GAS promoter.
[0195] To assess the effect of photoperiod on StBEL11 and StBEL29
mobility, levels of transgenic RNAs were measured in stolon tips of
GAS:StBEL11 and GAS:StBEL29 lines grown under either LD or SD
conditions using RTqPCR (FIG. 12). In all four transgenic lines for
StBEL11 and StBEL29, more transgenic RNA arising from the leaf
accumulated in stolon tips from plants grown under short-days than
long-days. These results suggest that movement of the transgenic
StBEL11 and StBEL29 RNAs is enhanced under SD conditions.
Consistent with StBEL5 RNA (Cho et al., "Polypyrimidine
Tract-Binding Proteins of Potato Mediate Tuberization Through an
Interaction With StBEL5 RNA," J. Expt. Bot. 66:6835-47 (2015),
which is hereby incorporated by reference in its entirety),
however, enhanced stability mediated by SDs may also contribute to
these steady-state level increases. Whereas shoot fresh weight from
these GAS lines was generally not affected by the enhanced levels
of StBEL11 and StBEL29 RNA (FIG. 13A), tuber growth was
significantly reduced in these lines grown under SD conditions
(FIG. 13B; FIG. 14). This reduction appears to be positively
correlated with the efficient movement and stability of both RNAs
into stolons under SD conditions (FIG. 12).
Example 10--Target Gene Activity
[0196] Since GAS:StBEL11 and StBEL29 lines exhibited a reduction in
tuber yield (FIG. 13B), the activity of select target genes was
measured in tuberizing stolons of these transgenic lines. Although
accumulation of RNAs for StSP6A and StPIN1 was enhanced in a
GAS:StBEL5 line relative to the WT line (Sharma et al., "Targets of
the StBEL5 Transcription Factor Include the FT Ortholog StSP6A,"
Plant Physiol. 170:310-24 (2016), which is hereby incorporated by
reference in its entirety), levels of RNA for these same marker
genes were reduced significantly for both GAS:StBEL11 and StBEL29
transgenic lines (FIGS. 15A, 15B). Both of these genes contain a
tandem TTGAC element within 2.0 kb (from the start codon) of their
upstream sequence, and both are strongly induced by StBEL5 activity
(Hannapel et al., "Phloem-Mobile Messenger RNAs and Root
Development," Front. Plant. Sci. 4:257 (2013) and Sharma et al.,
"Targets of the StBEL5 Transcription Factor Include the FT Ortholog
StSP6A," Plant Physiol. 170:310-24 (2016), which are hereby
incorporated by reference in their entirety). The suppressive
effect of StBEL11 and StBEL29 on these target genes appears not to
be mediated by a reduction in levels of StBEL5 activity. In an
independent experiment examining stolons of GAS:StBEL11 and StBEL29
lines, StBEL5 RNA levels were essentially unchanged or increased
only in transgenic line G:B11b (FIG. 16).
Example 11--StBEL11 and StBEL29 Function Antagonistically to
StBEL5
[0197] Potato tuberization is controlled by signals that arise from
the leaf under inductive conditions and are transported underground
via the sieve element system to activate cell growth in the stolon
meristem (Abelenda et al., "Flowering and Tuberization: A Tale of
Two Nightshades," Trends Plant Sci. 19:115-22 (2014), which is
hereby incorporated by reference in its entirety). Because of their
transport capacity, the search for these activating signals has
focused on primary products like miRNAs, full-length mRNAs, and
less abundant proteins that move through phloem cells in a
basipetal direction. In addition to full-length mRNAs, like StBEL5,
other prominent mobile tuberization signals have been identified.
These include like the FT-ortholog, StSP6A, a key regulator of
tuberization (Navarro et al., "Control of Flowering and Storage
Organ Formation in Potato by FLOWERING LOCUS T," Nature 478:119-22
(2011), which is hereby incorporated by reference in its entirety).
StSP6A protein accumulates in stolons of plants grown under SD and
its expression is closely correlated with tuber formation (Navarro
et al., "Control of Flowering and Storage Organ Formation in Potato
by FLOWERING LOCUS T," Nature 478:119-22 (2011) and Gonzalez-Schain
et al., "Potato CONSTANS is Involved in Photoperiodic Tuberization
in a Graft-Transmissible Manner," Plant J. 70:678-90 (2012), which
are hereby incorporated by reference in their entirety).
[0198] Two important miRNAs, miR172 and miR156, have also been
implicated in potato development (Martin et al.,
"Graft-Transmissible Induction of Potato Tuberization by the
MicroRNA miR172," Development 136:2873-81 (2009) and Bhogale et
al., "MicroRNA156: A Potential Graft-Transmissible MicroRNA That
Modulates Plant Architecture and Tuberization in Solanum tuberosum
ssp. andigena," Plant Physiol. 164:1011-27 (2014), which are hereby
incorporated by reference in their entirety). Through transcript
profiling of phloem sap, it is now known that hundreds of
full-length mRNAs are present in the sieve element system (Omid et
al., "Characterization of Phloem-Sap Transcription Profile in Melon
Plants," J. Exp. Bot. 58:3645-56 (2007); Deeken et al.,
"Identification of Arabidopsis thaliana Phloem RNAs Provides a
Search Criterion for Phloem-Based Transcripts Hidden in Complex
Datasets of Microarray Experiments," Plant J. 55:746-59 (2008);
Kehr et al, "Long Distance Transport and Movement of RNA Through
the Phloem," J. Exp. Bot. 59:85-92 (2008); and Notaguchi et al.,
"Identification of mRNAs That Move Over Long Distances Using an
RNA-Seq Analysis of Arabidopsis/Nicotiana benthamiana
Heterografts," Plant Cell Physiol. 56:311-21 (2015), which are
hereby incorporated by reference in their entirety).
[0199] Despite these insights, however, only a limited number of
RNAs have been confirmed to move and even fewer have been
associated with a phenotype. This latter group includes StBEL5
(Banerjee et al., "Dynamics of a Mobile RNA of Potato Involved in a
Long-Distance Signaling Pathway," Plant Cell 18:3443-57 (2006),
which is hereby incorporated by reference in its entirety) and
POTH1 (Mahaj an et al., "The mRNA of a Knotted1-Like Transcription
Factor of Potato is Phloem Mobile," Plant Mol. Biol. 79:595-608
(2012), which is hereby incorporated by reference in its entirety)
of potato, CmGAI of pumpkin (Haywood et al., "Phloem Long-Distance
Trafficking of GIBBERELLIC ACID-INSENSITIVE RNA Regulates Leaf
Development," Plant J. 42:49-68 (2005), which is hereby
incorporated by reference in its entirety), PFP-LeT6 from tomato
(Kim et al., "Developmental Changes Due to Long-Distance Movement
of a Homeobox Fusion Transcript in Tomato," Science 293:287-89
(2001), which is hereby incorporated by reference in its entirety),
and AUX/IAA (Notaguchi et al., "Phloem-Mobile Aux/IAA Transcripts
Target to the Root Tip and Modify Root Architecture," J. Int. Plant
Biol. 54:760-72 (2012), which is hereby incorporated by reference
in its entirety) and FLOWERING LOCUS T and CENTRORADIALIS (Li et
al., "Mobile FT mRNA Contributes to the Systemic Florigen
Signalling in Floral Induction," Sci. Rep. 1:73 (2011); Huang et
al., "Arabidopsis CENTRO-RADIALIS Homologue Acts Systemically to
Inhibit Floral Initiation in Arabidopsis," Plant J. 72:175-84
(2012); and Lu et al., "Long-Distance Movement of Arabidopsis
FLOWERING LOCUS T RNA Participates in Systemic Floral Regulation,"
RNA Biol. 9(5):653-62 (2012), which are hereby incorporated by
reference in their entirety) from Arabidopsis.
[0200] A recent report by Calderwood et al., "Transcript Abundance
Explains mRNA Mobility Data in Arabidopsis thaliana," Plant Cell
28:610-15 (2016), which is hereby incorporated by reference in its
entirety, indicated that movement of RNAs from phloem cells can be
explained by transcript abundance and RNA stability. This study
suggests that most of the identified transcripts that move from
companion cells into sieve elements do so via non-sequence-specific
transport. Whereas this study certainly establishes a strong case
for a non-specific mechanism controlling RNA movement, there is
also evidence that conserved RNA sequences that interact with
specific RNA-binding proteins may mediate transcript mobility (Ham
et al., "A Polypyrimidine Tract Binding Protein, Pumpkin RBP50,
Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex,"
Plant Cell 21:197-215 (2009) and Cho et al., "Polypyrimidine
Tract-Binding Proteins of Potato Mediate Tuberization Through an
Interaction With StBEL5 RNA," J. Expt. Bot. 66:6835-47 (2015),
which are hereby incorporated by reference in their entirety).
[0201] Because of the critical role that the polypyrimidine
tract-binding (PTB) proteins play in controlling StBEL5 transcript
movement and stability (Cho et al., "Polypyrimidine Tract-Binding
Proteins of Potato Mediate Tuberization Through an Interaction With
StBEL5 RNA," J. Expt. Bot. 66:6835-47 (2015), which is hereby
incorporated by reference in its entirety), future work will be
necessary to elucidate the processes that regulate mobility and
stability for StBEL11 and StBEL29. Evidence for a more specific
process is suggested, however, in the movement assays of this
study. Using heterografts and the same source promoter, StBEL11 and
StBEL29 RNAs moved liberally across the graft union into both roots
and stolons, whereas, movement of GUS transcripts was not detected
(FIGS. 11A-11C).
[0202] Whereas StBEL5 has been proposed to function as a mobile RNA
signal in potato that activates tuber growth (Banerjee et al.,
"Dynamics of a Mobile RNA of Potato Involved in a Long-Distance
Signaling Pathway," Plant Cell 18:3443-57 (2006) and Lin et al.,
"The Impact of the Long-Distance Transport of a BEL1-Like mRNA on
Development," Plant Physiol. 161:760-72 (2013), which are hereby
incorporated by reference in their entirety), here it is reported
that the phylogenetically-related StBELs, StBEL11 and StBEL29, are
also phloem-mobile, but act in opposition to StBEL5. Functional
antagonism has been reported previously among the BEL1-like TFs,
ARABIDOPSIS THALIANA HOMEOBOX 1, PENNYWISE and POUNDFOOLISH, in the
maintenance of the SAM and in the control of flowering time
(Rutjens et al., "Shoot Apical Meristem Function in Arabidopsis
Requires the Combined Activities of Three BEL1-Like Homeodomain
Proteins," Plant J. 58:641-54 (2009), which is hereby incorporated
by reference in its entirety).
[0203] Despite their antagonistic relationship, however, StBEL5,
StBEL11 and StBEL29 share a number of common features that are
unique among StBEL family members. All three exhibit RNA
accumulation and promoter activity associated with phloem cells
(Table 2; FIGS. 8A-8D; Banerjee et al., "Dynamics of a Mobile RNA
of Potato Involved in a Long-Distance Signaling Pathway," Plant
Cell 18:3443-57 (2006); Yu et al., "Tissue Integrity and RNA
Quality of Laser Microdissected Phloem of Potato," Planta
226:797-803 (2007); and Lin et al., "Transcriptional Analysis of
Phloem-Associated Cells of Potato," BMC Genom. 16:665 (2015), which
are hereby incorporated by reference in their entirety). Their RNAs
are ubiquitous throughout the plant and both movement and
accumulation of their RNAs are enhanced by a SD photoperiod. They
are the only BELs of potato with transcript levels consistently
affected by SDs in several organs (Chen et al., "Interacting
Transcription Factors From the Three Amino Acid Loop Extension
Superclass Regulate Tuber Formation," Plant Physiol. 132:1391-1404
(2003) and Sharma et al., "The BEL1-Like Family of Transcription
Factors in Potato," J. Expt. Bot. 65:709-23 (2014), which are
hereby incorporated by reference in their entirety), and all three
exhibit enhanced accumulation of their transcripts in stolons under
SD conditions.
[0204] This relationship with photoperiod suggests that their
movement and stability could be controlled by a common factor.
Recent work on the mobility of StBEL5 has shown that its RNA
interacts with RNA-binding proteins from the PTB family, StPTB1 and
StPTB6 (Cho et al., "Polypyrimidine Tract-Binding Proteins of
Potato Mediate Tuberization Through an Interaction With StBEL5
RNA," J. Expt. Bot. 66:6835-47 (2015), which is hereby incorporated
by reference in its entirety). This binding occurs on conserved
cytosine/uracil motifs present in the 3' UTR of StBEL5 and
facilitates stability as well as transport (Cho et al.,
"Polypyrimidine Tract-Binding Proteins of Potato Mediate
Tuberization Through an Interaction With StBEL5 RNA," J. Expt. Bot.
66:6835-47 (2015), which is hereby incorporated by reference in its
entirety). Similar motifs have been identified in the UTRs of
StBEL11 and StBEL29 that may facilitate binding to the StPTB
proteins. In over-expression lines of StPTB1 and StPTB6, movement
of StBEL11 and StBEL29 from leaves to stolon tips was enhanced
(FIGS. 17A-17D).
[0205] Polysomal RNA levels for StBEL11 and StBEL29 were positively
correlated with overall RNA accumulation, suggesting functional
activity of these TFs in those organs where they accumulate. In all
three of these StBEL TFs, the effect on growth appears to be
mediated by a similar set of target genes. For example, all three
regulate StSP6A activity (FIGS. 5A-5C; FIGS. 15A-15B; Sharma et
al., "Targets of the StBEL5 Transcription Factor Include the FT
Ortholog StSP6A," Plant Physiol. 170:310-24 (2016), which is hereby
incorporated by reference in its entirety). StBEL11 and StBEL29
appear to work on downstream target genes and have minimal effect
on StBEL5 transcription (FIG. 16). StBEL5 functions upstream of a
plethora of targets and induces the expression of six StBEL genes
in stolons, including StBEL11 and StBEL29, and numerous genes
involved in hormone metabolism (Sharma et al., "Targets of the
StBEL5 Transcription Factor Include the FT Ortholog StSP6A," Plant
Physiol. 170:310-24 (2016), which is hereby incorporated by
reference in its entirety; FIG. 18). Cross-regulation by StBEL5
contributes to inducing the expression of StBEL11 and StBEL29 in
stolons and other organs. In this way, all three may function
jointly to maintain a balance of controlled, localized cell growth
in these organs. The repressive transcription function observed for
StBEL11 and StBEL29 could be the result of a unique BEL/KNOX
interaction or it could be the effect of a novel sequence motif in
their proteins that modulates critical structural dynamics. In an
amino-acid sequence alignment of StBEL5, StBEL11, and StBEL29,
eleven unique amino acid runs of eight or more residues were
identified (FIG. 19). Among all three, there are very few sequence
differences within the highly conserved protein/protein domains
(BELL and SKY box) and the homeodomain.
Example 12--An Activator/Inhibitor Module for Tuberization
[0206] A system of activation and repression of growth is
consistent with the development of a new tuber from the stolon tip.
At the onset of tuber induction, the shoot apex ceases to elongate
and growth is initiated in a specific layer of cells within the
pith and cortex, resulting in swelling in the stolon tip that
spreads throughout the subapical portion of the meristem (Xu et
al., "Cell Division and Cell Enlargement During Potato Tuber
Formation," J. Exp. Bot. 49:573-82 (1998), which is hereby
incorporated by reference in its entirety). Further cell growth
arises from cells between the pith and cortex designated the
perimedullary zone just below the stolon apex and in close
proximity to vascular tissue (Xu et al., "Cell Division and Cell
Enlargement During Potato Tuber Formation," J. Exp. Bot. 49:573-82
(1998), which is hereby incorporated by reference in its entirety).
The orientation of cell division changes from transverse to
longitudinal leading to radial expansion. Most of the cell growth
occurs in this localized sub-apical region of the stolon meristem
(Xu et al., "Cell Division and Cell Enlargement During Potato Tuber
Formation," J. Exp. Bot. 49:573-82 (1998), which is hereby
incorporated by reference in its entirety) and changes in levels of
hormones like gibberellins, auxin, and cytokinins play pivotal
roles in regulating growth at this site (Xu et al., "The Role of
Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato
Tuber Formation In Vitro," Plant Physiol. 117:575-84 (1998), which
is hereby incorporated by reference in its entirety).
[0207] Growth below the apex creates a strong sink that
subsequently accumulates storage proteins and large amounts of
starch transported as sucrose via the phloem system. Control of
these processes includes both activation and suppression of the
growth of specific cell types unique to the tuberization program.
Mobile TFs that are transported through sieve elements and can move
cell-to-cell via plasmodesmata in the form of a full-length mRNA
are ideal for the fine-tuning of cell growth and cell dormancy.
Because of the high bioenergetic cost of this developing sink, the
efficient coordination of cell growth is critical. Due to their
mobility and specificity, this tripartite StBEL module could
readily contribute to cell fate determination in the stolon apex
during the transition process from stolon to tuber. Because
StBEL11/29 function antagonistically to StBEL5 for tuberization, it
is possible that these StBEL types function at different
developmental stages or in different cell types during the
stolon-to-tuber transition.
[0208] There are other examples of activation/suppression systems
that regulate plant growth through maintenance of the apical
meristem. One of the most widely studied systems is an
activator/inhibitor process that controls flowering (Lifschitz et
al., "Florigen and Anti-Florigen: A Systemic Mechanism for
Coordinating Growth and Termination in Flowering Plants," Front.
Plant. Sci. 5:465 (2014), which is hereby incorporated by reference
in its entirety). Flowering locus T (FT) protein acts as a mobile
florigen signal that moves into the apex and interacts with the
basic leucine zipper transcription factor, FD, to induce flowering
(Abe et al., "FD, A bZIP Protein Mediating Signals From the Floral
Pathway Integrator FT at the Shoot Apex," Science 309:1052-56
(2005); Wigge et al., "Integration of Spatial and Temporal
Information During Floral Induction in Arabidopsis," Science
309:1056-59 (2005); Corbesier et al., "FT Protein Movement
Contributes To Long Distance Signaling In Floral Induction of
Arabidopsis," Science 316:1030-33 (2007); Jaeger & Wigge, "FT
Protein Acts as a Long Range Signal in Arabidopsis," Curr. Biol.
17:1050-54 (2007); and Mathieu et al., "Export of FT Protein From
Phloem Companion Cells is Sufficient for Floral Induction in
Arabidopsis," Curr. Biol. 17:1055-60 (2007), which are hereby
incorporated by reference in their entirety).
[0209] TERMINAL FLOWER 1-like (TFL1) proteins function as floral
inhibitors and are antagonistic to FT function (Shannon &
Meeks-Wagner, "A Mutation in the Arabidopsis TFL1 Gene Affects
Inflorescence Meristem Development," Plant Cell 3:877-92 (1991),
which is hereby incorporated by reference in its entirety). A
single amino acid change in the FT protein is sufficient to
transform its function from an activator to a repressor (Hanzawa et
al., "A Single Amino Acid Converts a Repressor to an Activator of
Flowering," Proc. Nat'l. Acad. Sci. U.S.A. 102:7748-53 (2005),
which is hereby incorporated by reference in its entirety). There
have even been reports that the mRNAs of FT and CENTRORADIALIS, a
TFL1 homologue in Arabidopsis, move long distance to the shoot apex
via the phloem system (Huang et al., "Arabidopsis CENTRO-RADIALIS
Homologue Acts Systemically to Inhibit Floral Initiation in
Arabidopsis," Plant J. 72:175-84 (2012) and Lu et al.,
"Long-Distance Movement of Arabidopsis FLOWERING LOCUS T RNA
Participates in Systemic Floral Regulation," RNA Biol. 9(5):653-62
(2012), which are hereby incorporated by reference in their
entirety). Another report, however, indicated that FT mRNA movement
is not required to induce flowering (Notaguchi et al.,
"Long-Distance, Graft-Transmissible Action of Arabidopsis FLOWERING
LOCUS T Protein to Promote Flowering," Plant Cell Physiol.
49:1645-58 (2008), which is hereby incorporated by reference in its
entirety).
[0210] In another example of an activator/repressor process that
balances growth, the homeodomain TF, WUSCHEL (WUS), functions to
maintain stem cells in the SAM in an undifferentiated state (Schoof
et al., "The Stem Cell Population of Arabidopsis Shoot Meristems is
Maintained by a Regulatory Loop Between the CLAVATA and WUSCHEL
Genes," Cell 100:635-44 (2000) and Fletcher, "Shoot and Floral
Meristem Maintenance in Arabidopsis," Annu. Rev. Plant Biol.
53:45-66 (2002), which are hereby incorporated by reference in
their entirety). CLAVATA3 (CLV3), a peptide ligand, controls the
size of the stem cell domain by repressing WUS ( adnikova &
Simon, "How Boundaries Control Plant Development," Curr. Opin.
Plant Biol. 17:116-25 (2014), which is hereby incorporated by
reference in its entirety). In turn, the LATERAL ORGAN BOUNDARIES
DOMAIN TF, LBD15, maintains the stem cell pool through upregulation
of WUS (Sun et al., "Arabidopsis ASL11/LBD15 is Involved in Shoot
Apical Meristem Development and Regulates WUS Expression," Planta
237:1367-78 (2013), which is hereby incorporated by reference in
its entirety). The WUS-CLV feedback system forms a self-correcting
mechanism for maintaining a constant number of stem cells and the
SAM size at the shoot apex.
[0211] A model for tuber formation is currently arising that places
StBEL5 upstream in a regulatory network involving hormonal
metabolism and transcriptional controls that mediate tuber
formation (Sharma et al., "Targets of the StBEL5 Transcription
Factor Include the FT Ortholog StSP6A," Plant Physiol. 170:310-24
(2016), which is hereby incorporated by reference in its entirety).
The observation that StBEL5 induces transcription of StSP6A,
whereas StBEL11, and StBEL29 suppress its expression, is consistent
with this premise. Overall, these data suggest that StBEL5,
StBEL11, and StBEL29 could function collectively as phloem-mobile
mRNA signals in a whole-plant network in potato that modulates
storage organ development through the processes of cell growth
activation and suppression in the subapical portion of the stolon
tip.
[0212] Although preferred embodiments are depicted and described in
detail herein, it will be apparent to those skilled in the relevant
art that various modifications, additions, substitutions, and the
like can be made without departing from the spirit of the invention
and these are therefore considered to be within the scope of the
invention as defined in the claims which follow.
Sequence CWU 1
1
5312718DNASolanum tuberosum L. 1tttaagaaaa tctctcactt tctctttctc
ccaattataa taagaaaact ttctttcctc 60cttgttttta tttttaaaaa aatatttcag
tttagtttat ggttgaagat atttgatata 120gccttcatat atgtcactca
tgttccatca tcagccaagt gttagaagtc actttcttta 180acaagatttt
cttgaaaaat atttaaaaaa ttgaactcca aaaaaaagaa aaaaaggagt
240gtagttttct tgattggttg tgaaatttat ggctatgtac tatcaaggag
gctcagaaat 300ccaagctgat ggtctgcaga cactttattt gatgaaccct
aactatatag gctacactga 360cacacatcat catcatcatc aacaccaaca
acaatcagcc aacatgtttt tcttgaattc 420tgtggcggcg gggaattttc
cccacgtgtc cctccctttg caagcacatg cgcaggggca 480cttggttgga
gtgcccctgc cagctggttt tcaagatcct aaccgccctt ccattcagga
540aattccgacc tctcatcatg gccttttatc gcgtttgtgg acttctggtg
accaaaatac 600ccctagaggt ggtggaggag gaggagaagg aaatggaagt
caatcacata taccgtcttc 660cacggtggtt tctcccaact caggtagtgg
gggaggcacc accacggact ttgcttccca 720attagggttc caaagaccgg
ggttggtgtc accaacacag gcgcaccatc aaggtctttc 780tctaagcctt
tctccacaac aacaaatgaa tttcaggtct agtcttccac tagaccaccg
840cgatatttca acaacaaatc atcaagttgg aatactatca tcatcaccat
taccatcacc 900aggaacaaat accaatcata ctcgaggatt aggggcatca
tcgtcttttt cgatttctaa 960tgggatgata ttgggttcta agtacctaaa
agttgcacaa gatcttcttg atgaagttgt 1020taatgttgga aaaaacatca
aattatcaga ggttggtgca aaggagaaac acaaattgga 1080caatgaatta
atatctttgg ctagtgatga tgttgaaagt agcagccaaa aaaatagtgg
1140tgttgaactt actacagctc aaagacaaga acttcaaatg aagaaagcaa
agcttgttag 1200catgcttgat gaggtggatc aaaggtatag acaataccat
caccaaatgc aaatgattgc 1260aacatcattt gagcaaacaa caggaattgg
atcatcaaaa tcatacacac aacttgcttt 1320gcacacaatt tcaaagcaat
ttagatgttt aaaagatgca atttctgggc aaataaagga 1380cactagcaaa
actttagggg aagaagagaa cattggaggc aaaattgaag gatcaaagtt
1440gaaatttgtg gatcatcatt tacgccaaca acgtgcacta caacaattag
ggatgatgca 1500aaccaatgca tggaggccac aaagaggttt gcccgaaaga
gcggtttcgg ttctccgtgc 1560ttggcttttc gagcattttc ttcatccgta
tcccaaagat tcagataaaa tcatgcttgc 1620taagcaaaca gggctaacaa
ggagccaggt atcaaattgg tttataaatg ctagagttag 1680actatggaag
ccaatggtag aagaaatgta catggaagaa gtgaagaaaa acaatcaaga
1740acaaaatatt gagcctaata acaatgaaat tgttggttca aaatcaagtg
ttccacaaga 1800gaaattacca attagtagca atattattca taatgcttct
ccaaatgata tttctacttc 1860caccatttca acatctccga cgggcggcgg
cggttcgatt ccggctcaga cggttgcagg 1920tttctccttc attaggtcat
taaacatgga gaacattgat gatcaaagga acaacaaaaa 1980ggcaagaaat
gagatgcaaa attgttcaac tagtactatt ctctcaatgg aaagagaaat
2040catgaataaa gttgtgcaag atgagacaat caaaagtgaa aagttcaaca
acacacaaac 2100aagagaatgt tattctctaa tgactccaaa ttacacaatg
gatgatcaat ttggaacaag 2160gttcaacaat caaaatcatg aacaattggc
aacaacaaca acaacttttc atcaaggaaa 2220tggtcatgtt tctcttactc
tagggcttcc accaaattct gaaaaccaac acaattacat 2280tggattggaa
aatcattaca atcaacctac acatcatcca aatattagct atgaaaacat
2340tgattttcag agtggaaagc gatacgccac tcaactatta caagattttg
tttcttgatg 2400atatatataa tttgcaggta aatcagcttg aaattacatc
atgaaaggcc ttgaataaaa 2460gaaggggagt tgagatctag tgatcatata
aatatgtata ggtagaaagt ttagttagta 2520tatataggtt atacttctag
tttcttaaat ggagatacaa tttttgttgt tatttttgta 2580ttgagataac
tagctagctt ggattattta aagttgttgc atgcaaccaa agaagaagaa
2640aaaataatct atatatgcaa actatagtat gttgtaaatt ttgtgcgtct
ttttgtttca 2700atttgcatat atgtaaac 271828025DNASolanum tuberosum L.
2ttttttttat gtatatatac atttgatgaa gataatgttc tcttaagtga aaatcttgct
60tttatcatta gttagtactt acaattcttt ctgtcttatt ttatatgata tttttttaaa
120tttagtttac cccgaaaata aatgatatgt ttttatatat ttaactaatt
caatttaact 180aattcaattt taaacttctt tgaatctcaa tcgaattgcc
tcatttttga gaaggagttc 240gatttcaaac ccagattcga tccactccaa
gaaaaagaga aaagaaaaac aaatcaacta 300cgaaccccca ccccacccca
ccccaccccc caccatcgga aaaagggtca taagtagaaa 360taaagaaaaa
ttgagggact tctagcaact aatgtaatca attatgtatt atatatggac
420ccaacaaatt ggtggaaaaa gacgtttcct catttttcat atatctatgg
cctacttcct 480ttaagttaat gttttttttc ttcatctaat tttaagtcga
gtatttattt tgagactcgg 540attaatttaa attgatgttt tcaggaaaat
ttatcaaaag tgaaaatcta acttattgag 600aattttctta tttgtatgat
ttaaatttgt aacctctaaa taaagatgaa aaatcttaat 660catttcatca
ttactcgtaa ttattttctt cttgttagtg ttcactatac tctctctttc
720tctctaaaga tatttttgaa aaaaaatatc taaattatgc cagcatcaaa
tcattttata 780atagtgaaat taagattggg tctatttatt ttttccatca
cacgtatgta gaacccccca 840cccccaccct cgccgccacc ccaccccctt
actatcgagt ttaactaata tttattagta 900taaaaattat atttatctgt
tataacaagt aaaatgtctt atttttaaaa ggataaaggt 960atgagaaata
tcccaacttt gatcggattt actgttgcga tactaaactt tcatgaggat
1020ctattacctc cttcgactat ttaataccgt atttttatcc ccctgaacta
tttaatattg 1080tattttaaag gtatatatga ttatatgtgc caacgtggac
acattactat ttataatttt 1140gcattatttt ttatgtccac gtggacaaat
atatatgttt aaaatacggt attaaatagt 1200ctagggagct aataggtcct
catgaaagtt tagtatcgca acaacaaatt cgatcaaagt 1260tgagatattt
ttcaggccct tatccctatt tttaaaattg aaagtttaca tttttatgaa
1320gggttaaaac atgtaacatc atttaggtaa cttgatatag tataaaaaat
tatttacatt 1380atatataaat taaattcatg attactaaaa gaattcaatc
atcaggtcat ctttatctat 1440gaaatgtttt atttgtaaaa ttacaaacct
cacatttaaa aaagtttatc tataaatata 1500tttttaaata accttcctga
taatgtaaaa atatttatac tgacgattct tactgatttt 1560ttttttactg
tgtttttgag gggtggggtg ggggtgaggg taagggggat atgttgggag
1620acttacacta aataaacatg tcttctttat tcatattccc ctttatgtgt
tgtggagttt 1680taagaaaatc tctcactttc tctttctccc aattataata
agaaaacttt ctttcttcct 1740tgtttttatt tttaaaaaaa tatttcagtt
tagtacatgg ttgaagatat ttgatatagc 1800cttcatatat gtcactcatg
tgagtacaac ttttctccat atatatcaaa atcaagattt 1860tcatagttga
gtgattaatt aattgtatat aactcatcat atattatttg aattttcttt
1920gttaaaaatg ttttctatct ttagggtatt gcatggattt attataattt
ttttctatct 1980tactttctaa tttcaggttc catcatcagc caagtgttag
aagtcacttt ctttaacaag 2040attttcttaa aaaatattta aaaacttgaa
ctccaaaaaa aagaagaaaa ggagtgtaat 2100tttcttgatt ggttgtgaaa
tttatggcta tgtactatca aggaggctca gaaatccaag 2160ctgatggtct
gcagacactt tatttgatga accctaatta tataggctat actgacacac
2220atcatcatca tcaacaacac caacaacaat cagccaacat gtttttcttg
aattctgtgg 2280cggcggggaa ttttccccac gtgtccctcc ctttgcaagc
acatgcgcag gggcacttgg 2340ttggagtgcc cctgccagct ggttttcaag
atcctaaccg cccttccatt ccggaaattc 2400cgacctctca tcatggcctt
ttatcacgtt tgtggacttc tggtgaccaa aataccccta 2460gaggtggtgg
aggaggagga gaaggaaatg gaagtcaatc acatataccg tcttccacgg
2520tggtttctcc caactcaggt agtgggggag gcaccaccac ggactttgct
tcccaattag 2580ggttccaaag accggggttg gtgtcaccaa cacaggcgca
ccatcaaggt ctttctctaa 2640gcctttctcc acaacaacaa atgaatttca
ggtctagtct tccactagac caccgcgata 2700tttcaacaac aaatcatcaa
gttggaatac tatcaccatc accattacca tcaccaggaa 2760caaataccaa
tcatactcga ggattagggg catcatcgtc tttttcgatt tctaatggga
2820tgataatggg ttctaagtac ctaaaagttg cacaagatct tcttgatgaa
gttgttaatg 2880ttggaaaaaa catcaaatta tcagagggtg gtgcaaagga
gaaacacaaa ttggacaatg 2940aattaatctc tttggctagt gatgatgttg
aaagtagcag ccaaaaaaat attgttgttg 3000aacttactac agctcaaaga
caagaacttc aaatgaagaa agccaagctt gttagcatgc 3060ttgatgaggt
atatatactt ctaattattc atatattaat taattaatca tatatatata
3120ttaatcaaat tattcatata ttaattaatt aatcaatacc aagtttcttg
atttggagtt 3180tgatcattta ggcaaatttc actactatat ataaaacaca
aattctaacg gatgtttggt 3240cagtaagaaa ttctcaattt tcaatcaaac
atctattaga atttcacgtt ttttagtagt 3300gaaattaatt aaataatcta
aaaattgttc aatcgaattt acaacaaaac atccattgaa 3360attttacttt
cttttcaata gcgaaaccaa ttaaatagga ttgactaccc aattaatagt
3420tattatctta tcttcttctt gatttcaatc ttttttcaat aaaaagagta
attttaatta 3480tgagataata aactactgat tagttatgac aatctgaaaa
atcaactcct attaaatgat 3540ccaaaaagtg tacaaatttg tatatcttaa
tgttaatttc attgttttat ttatttattt 3600agttgctttt ttttccttct
tgggaagggg gaggggtcaa gttgctattg attcatatac 3660tagcaataat
tattgattta tttcaaaggt acaattttgt tcatcatgaa actataagct
3720agacaatata tgtggttcta agcttttttc tattgggggt ccaactaaag
ttaaagataa 3780tacacctaat atgtcttgtt gagttgacaa aaaatcaaag
gcacgtggtc ttattcaatc 3840actttattag aacctttcaa atttggaaat
attcttacct ttcttttgag ataatcacat 3900aaaaataaac ctttggaata
atttatattt ttggtattcc tattgatatt tgatatctgt 3960tttgaagctt
aactaatata aatatgcgtc gaaaagttat ctcactttag gagattaaaa
4020tgcttcttag taaaagtgac ttcatattca ggactcgaat aatattactt
gatgatcatt 4080tggacacaat tgatcaattg ggaaataatg aaatattatt
ttgcaaatca agttttattt 4140tataatttca ttagttattt cgacttgaac
ttgaaataaa gaatctgaag tttgaaaaat 4200tgatttaaag agtatttttt
tccactctaa gaacttcaaa caatttcaac ttcaacttca 4260tataatcata
ttttttttca acttcaatca gatatcgtcg tgatatgatc aatatcaatc
4320tacttatttt tattttattg tttgtatttt tttttgaatt ttttataggt
ggatcaaagg 4380tatagacaat accatcacca aatgcaaatg attgcaacat
catttgagca aacaacagga 4440attggatcat caaaatcata cacacaactt
gctttgcaca caatttcaaa gcaatttaga 4500tgtttaaaag atgcaatttt
tgggcaaata aaggacacaa gtaaaacttt aggggaagaa 4560gagaacattg
gaggcaaaat tgaaggatca aagttgaaat ttgtggatca tcatttacgc
4620caacaacgtg cactacaaca attagggatg atgcaaacca atgcatggag
gccacaaaga 4680ggtttgcccg aaagagcggt ttcggttctc cgcgcttggc
ttttcgagca ttttcttcat 4740ccgtaagtat ttgttgaaga cataattaag
taaattaata tgcatgtctt ttaatagttt 4800aagattttaa acaaagcaat
cacaacatcc tacatgtttc accgcttgtt ctccttatta 4860ggaaaaataa
ccaattgttc tagagtatat gagaaagaat cagactcgca atctagcatt
4920tgaagtggca aatacaagac taattaagta aatacaattt ttttttttaa
aataacagtt 4980taaacttttg aatgagatag atttaattaa caccttatat
tacctataag aaatgaactt 5040caatctctat ttttttttta aaaacaattt
tatacaccat gtagaaacct ttataaagaa 5100attaaattaa atcactcata
ccatttcttt taaatttcaa taaataaatt atatatttct 5160tgtcttgcag
gtatcccaaa gattcagata aaatcatgct tgctaagcaa acagggctaa
5220caaggagcca ggttcttgaa aaattcatca tctcaattta tatgacgcat
tttttaacat 5280atctaaaaaa gacgttttat ttctaattta gaaacaataa
aattttaaaa ttctcaacaa 5340tcatagtacc tctctatata actgtaacaa
catcttatta taacaaccaa ttttttgaat 5400atgccgtaaa aaagacatta
tattttcaat ttaggaacaa tataacttta aaattctcaa 5460caatcatagt
acctctctat ataactgtaa caacatctta ttataacaac caattttttg
5520aatatgccgt aaaaaagaca ttatattttc aatttaggaa caatataact
ttaaaattct 5580caacaatcat agtacctctc tatataacag taacaacatc
ttgttataac aaactaagtt 5640cttttttaaa ccaactttca ctacaacaaa
gataactttt agcggcaata tacatattaa 5700taaagaatac taaagctttt
accggcatta gttaatttca ttggatccat tatcgctata 5760gactgtagat
acatttacaa aaagtattaa ttaccactaa aaacacatat gtagtggcaa
5820ttttgctatt gctattaatt aattaatgtt ataaatataa tttttaatgt
agtgtttcat 5880gttatgttaa agtcatgtag tatatgttct ctacgaataa
tatttcacta tatcagacaa 5940aaaatatcta gaataaacaa tgatgttata
gaaagatttg acagcaagtc acacaaatat 6000gtacttaaga gtacttattt
tagactacaa gttttaaaag tcgatcgtct gttctttctt 6060aaaatacttt
tgaaaatgca ggtatcaaat tggtttataa atgctagagt tagactatgg
6120aagccaatgg tagaagaaat gtacatggaa gaagtgaaga aaaacaatca
agaacaaaat 6180attgagccta ataacaatga aattgttggt tcaaaatcaa
gtgttccaca agagaaatta 6240ccaattagta gcaatattat tcataatgct
tctccaaatg atatttctac ttccaccatt 6300tcaacatctc cgacgggtgg
cggcggttcg attccggctc agacggttgc aggttagttg 6360gaatataaag
aaagtcattt taaaagttgt cgttgtttga cctataatag gttttgagtc
6420gtggaaggca tcactaattt gcataaaggt aggttgtccc cttggggtat
ggtcttttca 6480tggagtaacg gtagagttgt ttccactgac ctatataagt
tacaggttcg agttgtggaa 6540ttggttgcgt tgttgatgct catgtcgggg
tagactgtct acaacacaca ccttgagata 6600caacctttta ttgtacccta
catgaatgtg aaatacttca cgcaccaaac tgcctaataa 6660ctcttagaaa
agaacacact tgactcacac atctatatat ctacgtagta cctcaattga
6720taaataatct aggatgatta gatggttaca catatcaaac atataatagg
ttcaagtcgt 6780agaaggcgtc actaatttgc atcaaggtag gttgtcccct
tggggtatga tcctttcatg 6840gaccatgtag acactcttga agttgagtct
tgaagtaaca gtaaagtcat ctccacgtga 6900cctatatata taagtcacaa
cttcgagttg tggagttggc taggctctca tcaggataga 6960ctgtcgacat
cacacttctt gaaatgcaac cttttttcga accttatgtg aatgtgagac
7020tacactcttg actaacatct atataactac tatacctcaa ttaataaaca
atctaggata 7080attagatggt ctcacactct aaacacctag gttagatcaa
aagacaataa aactagctag 7140agtacatttt tatttattgt aacaagtgtt
acttatcaaa gtgtgactct atattgttta 7200actaattaac atgtttaatt
tgtctaaaca ggtttctcct tcattaggtc attaaacatg 7260gagaacattg
atgatcaaag gaacaacaaa aaggcaagaa atgagatgca aaattgttca
7320actagtacta ttctctcaat ggaaagagaa atcatgaata aagttgtcca
agatgagaca 7380atcaaaagtg aaaagttcaa caacacacaa acaagagaat
gctattctct aatgactcca 7440aattacacaa tggatgatca atttggaaca
aggttcaaca atcaaaatca tgaacaattg 7500gcaacaactt ttcatcaagg
aaatggtcat gtttctctta ctctagggct tccaccaaat 7560tctgaaaacc
aacacaatta cattggattg gaaaatcatt acaatcaacc tacacatcat
7620ccaaatatta gctatgaaaa cattgatttt cagagtggaa agcgatacgc
cactcaacta 7680ttacaagatt ttgtttcttg atgatatata taatttccag
gtaaatcaac ttgaaattac 7740atcatgaaag gccttgaata aaagaagggg
agttgagatc tagtgatcat atatatatgt 7800ataggtagaa agtttagtta
gtatatatag gttatacttc tagtttctta aatggagata 7860caatttttgt
tgttgttttt gtattgagat aactagctag cttgggttat ttaaagttgt
7920tgcatgcaac caaagaagaa gaaaaaataa tctatatatg caaactatag
tatgttgtaa 7980attttgtgct tcttttaatt agtttcaatt tgcatatatg taaac
80253401DNASolanum tuberosum L. 3gaaatttatg gctatgtact atcaaggagg
ctcagaaatc caagctgatg gtctgcagac 60actttatttg atgaacccta actatatagg
ctacactgac acacatcatc atcatcatca 120acaccaacaa caatcagcca
acatgttttt cttgaattct gtggcggcgg ggaattttcc 180ccacgtgtcc
ctccctttgc aagcacatgc gcaggggcac ttggttggag tgcccctgcc
240agctggtttt caagatccta accgcccttc cattcaggaa attccgacct
ctcatcatgg 300ccttttatcg cgtttgtgga cttctggtga ccaaaatacc
cctagaggtg gtggaggagg 360aggagaagga aatggaagtc aatcacatat
accgtcttcc a 40142898DNASolanum tuberosum L. 4ttctttcttt ctttctcctc
tctctctctc taaaaagttg agtactttta ttagctctca 60tcacttcaca cagaagaaga
tggtattttt atttctttct gctgatggct gcatcaaatg 120atttgaaaag
ctgagtcaaa tcagaagaag aaaaagaaag ttataataat aataatgata
180atatcaaaaa tattattttc agattagttg gtgttatttg tttattgtgg
agaaaaaata 240aattaaaaag gaagaaaaaa tggcatctta ttttcatgga
aattcagaaa tacatgaagg 300aaatgatgga ttacaaactc taatactaat
gaatcctgga tatgttggat tttctgaaac 360acaacatcac cacgcgccac
cgccgccgcc gccaggtggc agcagcagca acatagtttt 420cttcaactcc
aatcctattg gaaattcaat gaacttatct cacgcgccac cacctcctcc
480accgcctcaa caacaattca tcggtatacc cctcgccacc gccgccttca
ccgccccatc 540ccaagactcc ggtaacaaca acaacaacga gtcaatctcc
gcccttcacg gcttcctagc 600tcgatcgtct cagtacgggt tttacaaccc
ggcaaacgac ctcacggcgg cgcgtgacgt 660cacacgcgct catcatcatc
atcagcagcc aagggctttc acttacctgt cctcgtccca 720gcagccgggg
tttgggaact tcacggcggc gcgtgagctt gtttcttcgc cttcgggttc
780ggcttcagct tcagggatac aacaacaaca acagcaacaa cagagtatta
gtagtgtgcc 840tttgagttct aagtacatga aggctgcaca agagctactt
gatgaagttg taaatgttgg 900aaaatcaatg aaaagtacta atagtactga
tgttgttgtt aataatgatg tcaagaaatc 960gaagaatatg ggcgatatgg
acggacagtt agacggagtt ggagcagaca aagacggagc 1020tccaacaact
gagctaagta caggggagag acaagaaatt caaatgaaga aagcaaaact
1080tgttaacatg cttgacgagg tggagcagag gtatagacat tatcatcacc
aaatgcagtc 1140agtgatacat tggttagagc aagctgctgg cattggatca
gcaaaaacat atacagcatt 1200ggctttgcag acgatttcga agcaatttag
gtgtcttaag gacgcgataa ttggtcaaat 1260acgatcagca agccagacgt
taggcgaaga agatagtttg ggagggaaga ttgaaggttc 1320aaggcttaaa
tttgttgata atcagctaag acagcaaagg gctttgcaac aattgggaat
1380gatccagcat aatgcttgga gacctcagag aggattgccc gaacgagctg
tttctgttct 1440tcgcgcttgg ctttttgaac atttcctcca tccttatccc
aaggattcag acaaaatgat 1500gctagcaaaa caaacaggac taactaggag
tcaggtgtcg aattggttca tcaatgctcg 1560agttcgtctt tggaagccaa
tggtggaaga gatgtacttg gaagagataa aagaacacga 1620acagaatggg
ttgggtcaag aaaagacgag caaattaggt gaacagaacg aagattcaac
1680aacatcaaga tccattgcta cacaagacaa aagccctggt tcagatagcc
aaaacaagag 1740ttttgtctca aaacaggaca atcatttgcc tcaacacaac
cctgcttcac caatgcccga 1800tgtccaacgc cacttccata cccctatcgg
tatgaccatc cgtaatcagt ctgctggttt 1860caacctcatt ggatcaccag
agatcgaaag catcaacatt actcaaggga gtccaaagaa 1920accgaggaac
aacgagatgt tgcattcacc aaacagcatt ccatccatca acatggatgt
1980aaagcctaac gaggaacaaa tgtcgatgaa gtttggtgat gataggcagg
acagagatgg 2040attctcacta atgggaggac cgatgaactt catgggagga
ttcggagcct atcccattgg 2100agaaattgct cggtttagca ccgagcaatt
ctcagcacca tactcaacca gtggcacagt 2160ttcactcact cttggcctac
cacataacga aaacctctca atgtctgcaa cacaccacag 2220tttccttcca
attccaacac aaaacatcca aattggaagt gaaccaaatc atgagtttgg
2280tagcttaaac acaccaacat cagctcactc aacatcaagc gtctatgaaa
ccttcaacat 2340tcagaacaga aagaggttcg ccgcaccctt gttaccagat
tttgttgcct gatcacaaaa 2400acaaaaacag gttttggcaa cagacaaact
tctgtcgcta aacaaggaca tgatttagcg 2460acagataact tcagtcgcta
acttagcgac tgaaaacttc tgtcgctaag catgaacatg 2520tattagcgac
atacagtatg caactgtatg tcactaaaca agaacatgat gaattagtga
2580cggacaactt ctgtcgctaa acaacaaaaa aaaatccatg ttttagtata
ttgtttctca 2640ttctatcata tcatggtagt gtaaagaatc aagaaacaag
ttttacatag taacagtctt 2700tatacattgg agatgaagaa ccatttaagt
tcttcaaaat agatagattt tctaggttac 2760ttctacaaga tatatatatg
gttgagggtt tgtatattaa ttttgggatt gttatattgg 2820atgtggaaaa
aaagtagtta ttttgggtgg tataaataaa ataatactcc atccatttta
2880gccaaaaaaa aaaaaaaa 289857921DNASolanum tuberosum L.
5tgagaagaaa acccaaagaa acttatgatt tataataaat tattagaaat ttctatggat
60ataaaatggt aaaaagtaag ttttattaaa tataaaaata tgtttttttt aatggaataa
120aaagcaaaaa aaaatcacat aaattagaat aaagatcgga gaaagtaaat
tataaataaa 180gacaagatga aaaacaaggc gataatgtaa atcatactaa
tcaatcgtta tacatattaa 240aaaatatcca gcgttacaac aacaaattta
acaatataat ataataaaat ttaactaaaa 300atcaaaataa aatgacattt
atcataacaa taattaacaa ccatccaaat atgatgtatg 360gataaaaggt
gaagagtatt agtatctttt gtttaaatct tatatattaa aattataaat
420ttaattatta ttttaaaaat tcttatataa attttaaatt ctgaatttgt
ccgacggcta 480atctaaagtc aaaagtaaat tttcataaat gtaggtccta
aattttttcc cacaattatc 540ttcttccaag ttgccaacac aaatcaataa
tgacaatagg gccctctccc ctatctcttc 600aaccctacct ctctttttct
ttctttatca cttcaagttc atatcatatt tcatactctc 660tcattttctt
ctggtctccg ttgtaattta tatgatatat tttttaatat ttaaaataat
720ttaattttaa attttttata ctctttaaaa
aattattata atcataagtt ataaaaaaaa 780ttaacttttt tttattcagt
caaatactat catataaatt aaaaaagaaa aagtatatgt 840taaatcctta
taattattat tgttaaagaa gaaaaaaggg aggttagtgg aagtggacgt
900tacctcgttt ttcatctgtc tgttttttct gacacacctt tgatctttga
tgatggatac 960gtcgctccgt tcatatttag gtgatactat attaatttca
agagttaaat aatgataaat 1020cacctaagac cgctaatgtt ccatctaatt
caagaacaag cccttctcaa tgtcttgcct 1080ttcgcatgtg ttttctttga
aattggaatt ccaaccaagt tcccttccca aagcgggaac 1140aagttggtgc
gaccgattaa agaagaagga caaagagtta aataatgaaa ttataattat
1200tttatattaa ttattataat ttataatatt ttttaaaaac taaatgttct
aatttaaagg 1260caaagtccaa atatttattt tataaatttt gaagcataat
tgggttttga ttaattattt 1320atatcaaatt aaatttattt taatacaaat
acataattta agacaaagct attgagttaa 1380agttatgtca aattaaatcc
gtaactttat aagctcaagg ggagaaagag agaaggattg 1440ttcattcctt
ataacgagtc tagagatctc atcctttatc gatgtaaggt tctttccatt
1500catcactccc ttgcgttaga accttttttt tttagactgg agcgtgcaca
ttcatggacc 1560attcttccca ttcgtcaatc cctcgtgtta gaatttttat
ttctcgaact agagtgtgtg 1620cattaacaga taccagatac cgatattttc
accctcattc aagccgtctc tggaagagct 1680atattggatg agcctgactt
tgataccata tcaaattaac tcttcaacct aattcataca 1740tcaaaagcta
gctcgcctta taagaagtct ttccattcgt cactccctcg tgttacaact
1800tacaagacta gctcaataaa aaattatcgt ccaaatttta taagaagtcc
attcatcaat 1860agcacctttc ctatttgtat ttgcacttaa aaaaaaaaag
gtgacttttg aaatttgaat 1920tatgccacat aaattatcct tcggtatagc
ccaatgattt gaccttggta ctttcatatt 1980ggaggtctca aatttgaaat
tccttaccag taaaaataaa aaatttacct tcctgaatcg 2040aacttatcgc
gccagacttc cttagacaca caaattagaa taaaaaaagt atattttatt
2100tttatatata agcaaaaaca cacactaact cacattcaca catccacatc
tttctttctt 2160tctttctcct ctctctctct ctaaaaagtt gagtactttt
attagctctc atcacttcac 2220acagaagaag atggtatttt tatttctttc
tgctgatggc tgcatcaaat gatttgaaaa 2280gctgagtcaa atcagaagaa
gaaaaagaaa gttataataa taataataat aatatcaaaa 2340atattatttt
caggtatggt acttctttac tcattaacaa tgtaaatata gaatttgaag
2400tttacgagct agttttctct tcttttttat tttgactagc agaaacagag
tcagagtcag 2460aatttgaagt ttataagtct tgaattctga ttttgtttga
gttcttgagt tctgaattga 2520taatttatac atgttgaatg aattttgtaa
gtatactttg aaacaaatct attgagttcg 2580attgaattca taaccgacac
tttagttccg ccacttttca gaggcggatc cagaatatga 2640aggttatgac
ttatgagtat tgtaaccttt tgagttactg aattctaaat taattttata
2700agtgagtaaa tacaaaattt gaaacaaaat tagctattga gttcagttga
attcgtataa 2760ccgacactct agctttgtca ctgctcaaag actgatctag
aatttgaagt ttatgagttt 2820tgaattctaa attgataatt tctacatgtt
agatggaatt tttaagataa atataaatta 2880aaattattga gttcgatcga
attcgatttg tgttttcctt tttcttcacc ttatttatca 2940agagaaatta
ttttaatttt tttttatcat tactgattca taaatctata tagatatata
3000tagatggata cattagagtt cctaaaaaat gttataaaga gtattttgtt
tttccctttc 3060ttgatttttt ttcgaaacta agatttcaat tttatcattt
ctgaatttta taacaacgat 3120tatcatagaa ttctaaattt actagttata
catatttatt taacgaatta ttaacataaa 3180tgcattattt gaataaaatt
tattagattt gaccgaactc gtatatgaac tttctctctc 3240atgtaatttc
agccgtaact gtgtgtattt cctttctctc tctaaaaaat ctgttatggt
3300gttctgtgtc actctaacaa aaaataaatt attcttcatt tcttccattg
tcagattata 3360atacaccacg tgcacttaca aattttgtga aaaccatatt
ttaatttaca aatccttttc 3420ataattcttt tttttaatca agaaaattaa
atttaattac attaagtatt ttttacaata 3480atattaacat attactaaat
aaattttcag gtttatttta ccatttttgt gattttgaag 3540tgttacaaag
tgtgaatggg ttggactttt tggacctcag ctaggtagct tcttgtcttc
3600aacacaaaag gtacatataa aaattacaat aaaattataa ccacttttac
ttaggaattt 3660taccatattt aggtaaaaaa aataaatcac tcgcctagtc
gtaataatca gtagcagagc 3720tagaggaacg aaaggcttca tctgaatctt
ctttatctga aatcatactg tatataaggt 3780caaaattcat tttttatgaa
cacttttgat gaaaatcatg tctctgccac taaagtcgta 3840atttgtaggc
atcaacaata tgtatatata tatatatata gtgattattg attatatacg
3900gtatatattg gttaaaagtt ttgaaaaata aggattaaag ttaatttcca
ttgtgtgttt 3960tcttgggatc aggggcggag ctagataagt gtaaaaaggg
tttatctaga cttcttccga 4020ccaaaaatta tacttatata catatacata
gtagatactg aatcccttgc ttttttcgta 4080tatgtacttc cgcatatttt
aaatttcttt aatgaaaatt ctgactcata tactgtttga 4140atgtttttgt
atttaatata tgtatgtttt gcctttttat tttggaaaaa atgatatatg
4200tggacttact tgacttgact ttaacttatt tttttttatt tttcagatta
gttggtgtta 4260tttgtttata gtggagaaaa aataaattaa aaaagaagag
aaaatggcat cttattttca 4320tggaaattca gaaatacaag aaggaaatga
tggattacaa actctaatac taatgaatcc 4380tggatatgtt ggattttctg
aaacacaaca tcaccacgcg ccgccgccgc caggtggcag 4440cagcaacaac
atagttttct tcaactccaa tcctcttgga aattcaataa acttatctca
4500cgcgccacca cctccgccac cgccacaaca acatttcgtc ggtatacctc
tcgccaccgc 4560cgccttcacc gccccatccc aagactccgg taacaacaac
aacaacgagt caatctccgc 4620ccttcacggc ttcctagctc gatcgtctca
gtacgggttt tacaacccgg ctaacgacat 4680cacggcggcg cgtgaggtca
cacgcgctca tcatcagcag cagcaagggc tttcacttag 4740cctgtcctca
tcccagcagc ctgggtttgg gaacttcacg gcggcgcgtg agattgtttc
4800ttcgcctacg cgttcggctt cggcttccgg gatacaacaa caacaacagc
aacaacaaag 4860tattagtagt gtgcctttga gttctaagta catgaaggct
gcacaagagc tacttgatga 4920agttgtaaat gttggaaaat caatgagaag
tactaatagt actgaagttg ttgttaataa 4980tgatgtcaag aaatcgaaga
ttatgaccga tatggatgga cagatagatg gaggagcaga 5040caaagacgga
actccaacaa ctgagctaag taccgcagag aggcaagaaa ttcaaatgaa
5100gaaagcaaaa cttgttaaca tgcttgacga ggtaaccttg ttgtcttttt
ctcagtaatg 5160ttgttgcatt cgtgtcagat cagagtctta aaattagtca
atagaagaaa cttcatttcc 5220tcgagtacgt gtaattgtgg ccttttcgac
ttccaactag tatttacaat agtgcactct 5280acattgataa gcttgacgac
aagtaggcaa agcgatggcc ttgttggttg ttatagtttt 5340ttggttatgt
tgctcggact ctgcaaaatt attgtcatac tcaagtcaga ttctccaaaa
5400tgcactattt ttggagtatc cgacttgcag tctgacattt attttttccg
aagagtctga 5460gcaacatagg ttttcttggc tttccaagat agtaagagaa
tggtctctat caaaaaaagt 5520tacatcatat cattactgaa aataagagca
aaaaagtatc tgtcaaatga taaagaccag 5580aacttcaaaa ctgttacttt
cgtcagggca ctgtcttgac aattgtaaac aaaaaatgaa 5640agaatttttc
gaaaataatt tcttcgaaat ctttgatcta aagctaaata tcggttcgat
5700tttgggtgtt gttatatagg tggagcagag gtatagacat tatcatcacc
aaatgcagtc 5760agtgatacac tggttggagc aagctgctgg tattggatca
gcaagaacat atacagcatt 5820ggctttgcag acgatttcga agcaatttag
gtgtcttaag gacgcgataa ttggtcaaat 5880acgatcagca ggcaagacgt
taggcgaaga agatagtttg ggagggaaga ttgaaggttc 5940aaggcttaaa
tttgttgaca atcagctaag acagcaaagg gctttgcaac aattgggaat
6000gatccagcat aatgcttgga gacctcagag aggattgccc gaacgagctg
tttctgttct 6060tcgcgcttgg ctttttgaac atttcctcca tccgtaagca
cgaaacaacc ctttttcatc 6120agctatgttg ctcggacttt tcaaaaacgt
tgtcgcacca gtgttggatc ctcgcagaat 6180gcattgattt tttgaggatc
cgacacatac ctgacgatat ttttgaagag tctgaacaac 6240atagcttagt
taaaagtact gtattttgat atattgtggc aatttgtttt gtatagctat
6300cccaaggatt cagacaaaat gatgctagca aaacaaacag ggctaactag
gagtcaggtc 6360agtgatatct gataacaaca ttgtcatttt tgattctcga
gttgatttct cagatggtca 6420cttaactgta gttattatat cagaaagtcg
ccttacttca acaaagagag tgacattctg 6480agataataac tgtgagttga
gtgaccatct gagaaatcaa ctcttggatt ctccgttttt 6540ggtttttact
aagttttgtt tttggacaat tcaggtgtcg aattggttca tcaatgctcg
6600agttcgtctt tggaagccaa tggtggaaga gatgtacttg gaagagataa
aagaacagaa 6660cggattgggt caagaaaaga cgagcaaatt aggcgaacag
aacgaagatt caacaacatc 6720aagatccatt gctacacaag acaaaagccc
tggttcagat agccaaaaca agagttttgt 6780ctcaaaacag gacaatcatt
tgccccaaca caaccctgct tcaccaatgc cgatgtccaa 6840caccacttcc
atacctccta tcggtatgaa catccgtaat cagtctgctg gtttcaacct
6900cattggatca ccagagatcg aaagcatcaa cattactcaa gggagtccaa
agaaaccaag 6960gaacaacgag atgttgcatt caccaaacag cattccatcc
atcaacattg atgtaaagcc 7020taacgagcaa caaatgtcga tgaagtttgg
tgatgatagg caagacagag atggattctc 7080actaatggga ggaccgatga
acttcatggg aggattcgga gcctatccca ttggagaaat 7140tgctcggttt
agcaccgagc aattctcagc accatactca accagtggca cagtttcact
7200cactcttggc ctaccacata acgaaaacct ctcaatgtca gcaacacacc
acagtttcct 7260tccaattcca acacaaaaca tccaaattgg aagtgaacca
aatcatgagt ttggtagctt 7320aaacacacca acatcagctc actcaacatc
aagcgtctac gaaaatttca acattcagaa 7380cagaaagagg ttcgccgcac
ccttgttacc agattttgtt gcctgatcac aaaaacaaaa 7440acaggattta
gcgacagaca aacttctgtc gctaaacaag aacatgattt agcgacagat
7500aacttcagtc gctaacttag cgactgaaaa cttctgtcgc taaacatgaa
catgtattag 7560cgacatacag tatacaactg tatgtcgcta aacaagaaca
tgatgaatta gtgacggaca 7620acttctgtcg ctaaacaaca aaaaaagatc
catgttttag tatattgttt ctcattctat 7680catatcatgg tagtgtaaag
aatcaagaaa caagttttac atagttacat agtctttata 7740cattggagat
gaagaaccat ttaagttctt caaaatagat agattttcta ggttacttct
7800agaagatata tatatggttg agggtttgta tattaatttt gggattgtta
tattggatgt 7860ggaaaaaaag tagttatttt gggtggtata aataaaataa
tactccatcc attttagcca 7920a 79216799DNASolanum tuberosum L.
6gtgttatttg tttattgtgg agaaaaaata aattaaaaag gaagaaaaaa tggcatctta
60ttttcatgga aattcagaaa tacatgaagg aaatgatgga ttacaaactc taatactaat
120gaatcctgga tatgttggat tttctgaaac acaacatcac cacgcgccac
cgccgccgcc 180gccaggtggc agcagcagca acatagtttt cttcaactcc
aatcctattg gaaattcaat 240gaacttatct cacgcgccac cacctcctcc
accgcctcaa caacaattca tcggtatacc 300cctcgccacc gccgccttca
ccgccccatc ccaagactcc ggtaacaaca acaacaacga 360gtcaatctcc
gcccttcacg gcttcctagc tcgatcgtct cagtacgggt tttacaaccc
420ggcaaacgac ctcacggcgg cgcgtgacgt cacacgcgct catcatcatc
atcagcagcc 480aagggctttc acttacctgt cctcgtccca gcagccgggg
tttgggaact tcacggcggc 540gcgtgagctt gtttcttcgc cttcgggttc
ggcttcagct tcagggatac aacaacaaca 600acagcaacaa cagagtatta
gtagtgtgcc tttgagttct aagtacatga aggctgcaca 660agagctactt
gatgaagttg taaatgttgg aaaatcaatg aaaagtacta atagtactga
720tgttgttgtt aataatgatg tcaagaaatc gaagaatatg ggcgatatgg
acggacagtt 780agacggagtt ggagcagac 79972735DNASolanum tuberosum L.
7catgcagaga taaaaatata gatcagtctg acaagaaggc aacttctcaa agcttagaga
60gctaccaccc gaagatagac agttagttac atgtactgtt atagataaaa ggagaaatcc
120gaagaagaaa gaattttttt tgcagatatg tactatcaag gaacctcgga
taatactaat 180atacaagctg atcatcaaca acgtcataat catgggaata
gtaataataa taatattcag 240acactttatt tgatgaaccc taacaattat
atgcaaggct acactacttc tgacacacag 300cagcagcagc agttactttt
cctgaattct tcaccagcag caagcaacgc gctttgccat 360gcgaatatac
aacacgcgcc gctgcaacag cagcactttg tcggtgtgcc tcttccggca
420gtaagtttgc acgatcagat caatcatcat ggacttttac agcgcatgtg
gaacaaccaa 480gatcaatctc agcaggtgat agtaccatcg tcgacggggg
tttctgccac gtcatgtggc 540gggatcacca cggacttggc gtctcaattg
gcgtttcaga ggccgattcc gacaccacaa 600caccgacagc agcaacaaca
gcaaggcggt ctatctctaa gcctttctcc tcagctacaa 660cagcaaatta
gtttcaataa caatatttca tcctcatcac caaggacaaa taatgttact
720attaggggaa cattagatgg aagttctagc aacatggttt taggctctaa
gtatctgaaa 780gctgcacaag agcttcttga tgaagttgtt aatattgttg
gaaaaagcat caaaggagat 840gatcaaaaga aggataattc aatgaataaa
gaatcaatgc ctttggctag tgatgtcaac 900actaatagtt ctggtggtgg
tgaaagtagc agcaggcaga aaaatgaagt tgctgttgag 960cttacaactg
ctcaaagaca agaacttcaa atgaaaaaag ccaagcttct tgccatgctt
1020gaagaggtgg agcaaaggta cagacagtac catcaccaaa tgcaaataat
tgtattatca 1080tttgagcaag tagcaggaat tggatcagcc aaatcataca
ctcaattagc tttgcatgca 1140atttcgaagc aattcagatg cctaaaggat
gcaattgctg agcaagtaaa ggcgacgagc 1200aagagtttag gtgaagagga
aggcttggga gggaaaatcg aaggctcaag actcaaattt 1260gtggaccatc
atctaaggca acaacgcgcg ctgcaacaga taggaatgat gcaaccaaat
1320gcttggagac cccaaagagg tttacctgaa agagctgtct ctgtccttcg
tgcttggctt 1380ttcgagcatt ttcttcatcc ttacccaaag gattcagaca
aaatcatgct tgctaagcaa 1440acggggctaa caaggagcca ggtgtctaac
tggttcataa atgctcgagt tcgattatgg 1500aagccaatgg tagaagaaat
gtacttggaa gaagtgaaga atcaagaaca aaacagtact 1560aatacttcag
gagataacaa aaacaaagag accaatataa gtgctccaaa tgaagagaaa
1620catccaatta ttactagcag cttattacaa gatggtatta ctactactca
agcagaaatt 1680tctacctcaa ctatttcaac ttcccctact gcaggtgctt
cacttcatca tgctcacaat 1740ttctccttcc ttggttcatt caacatggat
aatactacta ctactgttga tcatattgaa 1800aacaacgcga aaaagcaaag
aaatgacatg cacaagtttt ctccaagtag tattctttca 1860tctgttgaca
tggaagccaa agctagagaa tcatcaaata aagggtttac taatccttta
1920atggcagcat acgcgatggg agattttgga aggtttgatc ctcatgatca
acaaatgacc 1980gcgaattttc atggaaataa tggtgtctct cttactttag
gacttcctcc ttctgaaaac 2040ctagccatgc cagtgagcca acaaaattac
ctttctaatg acttgggaag taggtctgaa 2100atggggagtc attacaatag
aatgggatat gaaaacattg attttcagag tgggaataag 2160cgatttccga
ctcaactatt accagatttt gttacaggta atctaggaac atgaatacca
2220gaaagtctcg tattgatagc tgaaaagata aaaggaagtt agggatactc
ttatattgtg 2280tgaggccttc tggcccaagt cggaggaccc aatttgatac
aacctatcat aggagaaaag 2340aagtggagac taaattaaag taacaaaatt
ttaaagcaca ctttctagta tatatacttc 2400ttttttttat agtatagaaa
agaagagatt ttgtgcttta gtgtatagat agagtctact 2460tagtataggt
tatacttcta gttccttgag aagattgata caactagtag tatttttttt
2520cttttgggtt ggcttggagt actattttaa gttattggaa actagctata
gtaaatgttg 2580taaagttgtg atattgttcc tctcaatttg catataattt
gaaatatttt gtacctacta 2640gctagtctct aaattatgtt tccattgctt
gtaattgcaa ttttatttga attttgtgct 2700atcattatta gattagcaaa
aaaaaaaaaa aaaaa 273584015DNASolanum tuberosum L. 8gtaggtacaa
aatatttcaa attatatgca aattgagagg aacaatatca caactttaca 60acatatacta
tagctagttt ccatattaac ttaaaatagt actccaagcc aacccagaaa
120gaaaaaaaat actactagtt gtatcaatct tctcaaggaa ctagaagtat
aacctatact 180aagtagactc tatctataca ctaaagcaca aaatctcttc
ttttctatac tataaaaaaa 240agaagtatat atactagaaa gtgtgcttta
aaattttgtt actttaattt tgtctccact 300tcttttctcc tatgataggt
tgtatcaaat tgggtcctcc gacttgggcc agaaggcctc 360acacaatata
agagtatccc taacttcctt ttatcttttc agctatcaat acgagacttt
420ctggtattca tgttcctaga ttacctgtaa caaaatctgg taatagttga
gtcggaaatc 480gcttattccc actctgaaaa tcaatgtttt catatcccat
tctattgtaa tgactcccca 540tttcaggcct acttcccaag tcattagaaa
ggtaattttg ttggctcact ggcatggcta 600ggttttcaga aggaggaagt
cctaaagtaa gagagacacc attatttcca tgaaaattcg 660cggtgatttg
ttgatcatga ggatcaaacc ttccaaaatc tcccatcgcg tatgctgcca
720ttaaaggatt agtaaaccct ttatttgatg attctctagc tttggcttcc
atgtcaacag 780atgaaagaat actacttgga gaaaacttgt gcatgtcatt
tctttgcttt ttcgcgttgt 840tttcaatatg atcaacagta gtagtagtag
tattatccat gttgaatgaa ccaaggaagg 900agaaattgtg agcatgatga
agtgaagcac ctgcagtagg ggaagttgaa atagttgagg 960tagaaatttc
tgcttgagta gtagtaatac catcttgtaa taagctgcta gtaataattg
1020gatgtttctc ttcatttgga gcctctttgt ttttgttatc tcctgaagta
ttagtactgt 1080tttgttcttg attcttcact tcttccaagt acatttcttc
taccattggc ttccataatc 1140gaactcgagc atttatgaac cagttagaga
cctgcatttt catatatatt aagaattttt 1200ttataaagaa aagaaaggaa
ttaatccaag aattatagta aaatgtgtgt ctaagaacct 1260ggctccttgt
tagccccgtt tgcttagcaa gcatgatttt gtctgaatcc tttgggtaac
1320tgcaatataa aaatatatta agaaaaaaaa aattatagtt aaaaacatac
tcctatatta 1380gagaagaatg ggcgaattca gaatttagaa taataatgtg
atcttattat atacatgaac 1440atattttatt ttttgtgtgt atgcatatat
agtttgagtt aaaagtaagt gtctttttaa 1500ccgtccaaac gaaagtttca
aaataactgg cgttgctcta aaaatcactt aatttgttcc 1560taatggatgg
acatgttaaa accatataaa agacacttag taaaatatag gacgacaggc
1620gtatatatga cgcaaaaatt ggttagaata atcaattttc tatctactac
tccgtagata 1680tttttcacat tgttaaattt ttcaaagaaa taaatagttt
aggagtactc acggatgaag 1740aaaatgctcg aaaagccaag cacgaaggac
agagacagct ctttcaggta aacctctttg 1800gggtctccaa gcatttggtt
gcatcattcc tagctgttgc agcgcgcgtt gttgccttag 1860atgatggtcc
acaaatttga gtcttgagcc ttcgattttc cctactaagc cttcctcttc
1920acctaaactc ttgctcgtcg cctttacttg ctcagcaatt gcatccttta
ggcatctgaa 1980ttgcttcgaa attgcatgca aagctaattg agtgtatgat
ttggctgaac caattcctgc 2040tacttgctca aatgatgata caattatttg
catttggtga tggtactgtc tgtacctttg 2100ctccacctga acaaaaaaaa
gggagtaata ttaaactttt accagtctgt cgttttacaa 2160catgaagtta
tcttatgttg gctactattg aaattaaaga attttatttc agttaaaaga
2220tcatatatat atatatatat atattccaag tgagaaataa attgagtagt
atattttgca 2280aaattttgta aaccaacgaa tttttgagag tcattagatt
gaggacacat ctgagtggac 2340attatgcgtg gtgtaaaaaa ggtgaataag
agatagtggt ttgaattttg gtgcagcgga 2400agacatttca ggttcgtagc
taactttggt gtattatctt tatagcttta gttggacccg 2460cagaagaaaa
tttaagagcc acacattgtc agtttgtttt aatatcaacg tacgtgattg
2520gtctcttgtt cttcaactaa ttaacaaaac ctgtacattt catttaccaa
ctactattgt 2580tgcaaacata tataaatcaa cagtttcatc cattcaattt
tttatgagaa aaattacagt 2640tttgaatcat ttgaaaataa aattttaaat
atatatgtcg aattcagtag ttttagtgtt 2700aagaatccga aattcataga
ctcaaaattc aggatcatac ctcttcaagc atggcaagaa 2760gcttggcttt
tttcatttga agttcttgtc tttgagcagt tgtaagctca atagcaactt
2820catttttctg cctgctgcta ctttcaccac caccaccacc accagaacta
ttagtgttga 2880catcactagc caaaggcatt gaattatcct tcttttgatc
atctcctttg atgctttttc 2940caacaatatt aacaacttca tcaagaagct
cttgtgcagc tttcagatac ttagagccta 3000aaaccatgtt gctagaactt
ccatctaatg ttcctctaat agtaacatta tttgtccttg 3060gtgatgagga
tgaaatattg ttattgaaac taatttgctg ttgttgctga ggagaaaggc
3120ttagagatag accgccttgc tgttgttgct gctgctgtcg gtgttgtggt
gtcggaatcg 3180gcctctgaaa cgccaattga gacgccaagt ccgtggtgat
cccgccacat gacgtggcag 3240aaacccccgt cgacgatggt actatcacct
gctgagattg atcttggttg ttccacatac 3300gctgtaaaag tccatgatga
ttgatctgat cgtgcaaact tactgccgga agaggcacac 3360cgacaaagtg
ctgctgttgc agcggcgcgt gttgtatatt cgcatggcaa agcgcgttgc
3420ttcctgctgg tgaagaattc aggaaaagta actgctgctg ctgctgtgtg
tcagaagtag 3480tgtagccttg catataattg ttagggttca tcaaataaag
cgtctgaata ttattattat 3540tactactatt cccatgatta tgatgttgtt
gatgatcagc ttgtatatta ttatccgagg 3600ttccttgata gtacatatct
gcaaaaaaaa atctttcttc ttcgaatttc tccttttatc 3660tacaacagta
cctgtaaaca gaaagtaaca aaggagaaaa ggcttcaaat aagtccacac
3720aaacattttt ataagtaaac ggaagggaat tctttatagt gaaaaattaa
attttgttta 3780cagagatctt caactataaa taaaaaaaac aggaaaatga
tataaaagaa agagaaagag 3840atgaaaggag ctttagcaaa aaaatcagtc
actcacacat acacacatgt aactaactgt 3900ctatcttcgg gtggtagctc
tctaagcttt gagaagttgc cttcttgtca gactgatcta 3960tatttttctc
tctgcattct catctcttca accacaaaaa ggaaatatga ataaa
4015929DNAArtificialSolanum tuberosum L. 9rmrbcatcta gagtaggggg
ggaggcacc 291036DNAArtificialSolanum
tuberosum L. 10rmrbrgagag ctcgaagcac aaaatttaca atatac
361133DNAArtificialSolanum tuberosum L. 11rmrbacatct agattagctc
tcatcacttc aca 331232DNAArtificialSolanum tuberosum L. 12rmrbragagg
tacctaccac ccaaaatact ac 321337DNAArtificialSolanum tuberosum L.
13rmrstbasga gctcgaaatt tatggctatg tactatc
371436DNAArtificialSolanum tuberosum L. 14rmrstbasrt ctagagtgga
agacggtata tgtgat 361537DNAArtificialSolanum tuberosum L.
15rmrstbasga gctcgtgtta tttgtttatt gtggaga
371635DNAArtificialSolanum tuberosum L. 16rmrstbasrt ctagagtctg
ctccaactcc gtcta 351734DNAArtificialSolanum tuberosum L.
17rmrbtataag cttaactaac taactaactg tccc 341834DNAArtificialSolanum
tuberosum L. 18rmrbrgagtc tagaactcca caacacataa aggg
341934DNAArtificialSolanum tuberosum L. 19rmrbcacaag ctttgagaag
aaaaccaaag aaac 342035DNAArtificialSolanum tuberosum L.
20rmrbraacgg atccagatgt ggatgtgtga atgtg 352148DNAArtificialSolanum
tuberosum L. 21rmrgasbtat attatatccc gggtttaaga aaatctctca ctttctct
482249DNAArtificialSolanum tuberosum L. 22rmrgasbrta tattatatga
gctcgtttac atatatgcaa attgaaaca 492348DNAArtificialSolanum
tuberosum L. 23rmrgasbtat attatatccc gggttctttc tttctttctc ctctctct
482449DNAArtificialSolanum tuberosum L. 24rmrgasbrta tattatatga
tatcggctaa aatggatgga gtattattt 492525DNAArtificialSolanum
tuberosum L. 25rmraggacac tagcaaaact ttagg
252625DNAArtificialSolanum tuberosum L. 26rmrrctttga ggcttccatg
cattg 252723DNAArtificialSolanum tuberosum L. 27rmrcatttgc
ctcaacacaa ccc 232825DNAArtificialSolanum tuberosum L. 28rmrrtgatgc
tttcgatctc tggtg 252926DNAArtificialSolanum tuberosum L.
29rmrgadhgaa ggactggaga ggtgga 263029DNAArtificialSolanum tuberosum
L. 30rmrgadhrga caacagaaac atcagcagt 293127DNAArtificialSolanum
tuberosum L.misc_feature(4)..(4)n is a, c, g, or t 31rmrntgcggg
actctaatca taaaaac 273228DNAArtificialSolanum tuberosum L.
32rmrgsgstgg aaacggcaga gaaggtac 283328DNAArtificialSolanum
tuberosum L. 33rmrgsattag tgacggacaa cttctgtc
283431DNAArtificialSolanum tuberosum L. 34rmrgsgtaaa tcagcttgaa
attacatcat g 313536DNAArtificialSolanum tuberosum L. 35rmrstbasrt
ctagagtgga agacggtata tgtgat 363630DNAArtificialSolanum tuberosum
L. 36rmrstbasrc agaaaatcca acatatccag 303731DNAArtificialSolanum
tuberosum L.misc_feature(4)..(4)n is a, c, g, or t 37rmrnstrscr
gcaacaggat tcaatcttaa g 313825DNAArtificialSolanum tuberosum
L.misc_feature(6)..(6)n is a, c, g, or t 38rmrkanrgga ttgcacgcag
gttct 253927DNAArtificialSolanum tuberosum L.misc_feature(6)..(6)n
is a, c, g, or t 39rmrkanrrcg tcaagaaggc gatagaa
274035DNAArtificialSolanum tuberosum L. 40rmrbmrtgsc tatatatgca
aactatagta tgttg 354136DNAArtificialSolanum tuberosum L.
41rmrbmrtgsc ttctagaaga tatatatatg gttgag
364239DNAArtificialSolanum tuberosum L.misc_feature(4)..(4)n is a,
c, g, or t 42rmrntrvctr sccgcaacag gattcaatct taagaaact
394330DNAArtificialSolanum tuberosum L. 43rmrstactrt ggaaaagctt
gcctatgtgg 304430DNAArtificialSolanum tuberosum L. 44rmrstactrt
rctgctcctg gcagtttcaa 304528DNAArtificialSolanum tuberosum
L.misc_feature(6)..(6)n is a, c, g, or t 45rmrstnrttc atctaaaggg
ccaacacc 284629DNAArtificialSolanum tuberosum
L.misc_feature(6)..(6)n is a, c, g, or t 46rmrstnrtrg ttgtatagct
ccccgctca 294734DNAArtificialSolanum tuberosum L. 47rmrstgartt
tctctacaat gagttcacat ggtc 344833DNAArtificialSolanum tuberosum L.
48rmrstgartr gggacaacct attatcacca agc 334932DNAArtificialSolanum
tuberosum L. 49rmrstaartc tgatcttcga tcaatttcat gg
325032DNAArtificialSolanum tuberosum L. 50rmrstaartr gacctattgc
tgccttgtgc ta 3251700PRTArtificialSolanum tuberosum L. 51Ser Leu
Ala Asn Met Thr Asx Glu Arg Ser Met Leu Met Tyr Tyr Gln1 5 10 15Gly
Thr Ser Asp Asn Thr Asn Ile Gln Ala Asp His Gln Gln Arg His 20 25
30Asn His Gly Asn Ser Asn Asn Asn Asn Ile Gln Thr Leu Tyr Leu Met
35 40 45Asn Pro Asn Asn Tyr Met Gln Gly Tyr Thr Thr Ser Asp Thr Gln
Gln 50 55 60Gln Gln Gln Leu Leu Phe Leu Asn Ser Ser Pro Ala Ala Ser
Asn Ala65 70 75 80Leu Cys His Ala Asn Ile Gln His Ala Pro Leu Gln
Gln Gln His Phe 85 90 95Val Gly Val Pro Leu Pro Ala Val Ser Leu His
Asp Gln Ile Asn His 100 105 110His Gly Leu Leu Gln Arg Met Trp Asn
Asn Gln Asp Gln Ser Gln Gln 115 120 125Val Ile Val Pro Ser Ser Thr
Gly Val Ser Ala Thr Ser Cys Gly Gly 130 135 140Ile Thr Thr Asp Leu
Ala Ser Gln Leu Ala Phe Gln Arg Pro Ile Pro145 150 155 160Thr Pro
Gln His Arg Gln Gln Gln Gln Gln Gln Gly Gly Leu Ser Leu 165 170
175Ser Leu Ser Pro Gln Leu Gln Gln Gln Ile Ser Phe Asn Asn Asn Ile
180 185 190Ser Ser Ser Ser Pro Arg Thr Asn Asn Val Thr Ile Arg Gly
Thr Leu 195 200 205Asp Gly Ser Ser Ser Asn Met Val Leu Gly Ser Lys
Tyr Leu Lys Ala 210 215 220Ala Gln Glu Leu Leu Asp Glu Val Val Asn
Ile Val Gly Lys Ser Ile225 230 235 240Lys Gly Asp Asp Gln Lys Lys
Asp Asn Ser Met Asn Lys Glu Ser Met 245 250 255Pro Leu Ala Ser Asp
Val Asn Thr Asn Ser Ser Gly Gly Gly Glu Ser 260 265 270Ser Ser Arg
Gln Lys Asn Glu Val Ala Val Glu Leu Thr Thr Ala Gln 275 280 285Arg
Gln Glu Leu Gln Met Lys Lys Ala Lys Leu Leu Ala Met Leu Glu 290 295
300Glu Val Glu Gln Arg Tyr Arg Gln Tyr His His Gln Met Gln Ile
Ile305 310 315 320Val Leu Ser Phe Glu Gln Val Ala Gly Ile Gly Ser
Ala Lys Ser Tyr 325 330 335Thr Gln Leu Ala Leu His Ala Ile Ser Lys
Gln Phe Arg Cys Leu Lys 340 345 350Asp Ala Ile Ala Glu Gln Val Lys
Ala Thr Ser Lys Ser Leu Gly Glu 355 360 365Glu Glu Gly Leu Gly Gly
Lys Ile Glu Gly Ser Arg Leu Lys Phe Val 370 375 380Asp His His Leu
Arg Gln Gln Arg Ala Leu Gln Gln Ile Gly Met Met385 390 395 400Gln
Pro Asn Ala Trp Arg Pro Gln Arg Gly Leu Pro Glu Arg Ala Val 405 410
415Ser Val Leu Arg Ala Trp Leu Phe Glu His Phe Leu His Pro Tyr Pro
420 425 430Lys Asp Ser Asp Lys Ile Met Leu Ala Lys Gln Thr Gly Leu
Thr Arg 435 440 445Ser Gln Val Ser Asn Trp Phe Ile Asn Ala Arg Val
Arg Leu Trp Lys 450 455 460Pro Met Val Glu Glu Met Tyr Leu Glu Glu
Val Lys Asn Gln Glu Gln465 470 475 480Asn Ser Thr Asn Thr Ser Gly
Asp Asn Lys Asn Lys Glu Thr Asn Ile 485 490 495Ser Ala Pro Asn Glu
Glu Lys His Pro Ile Ile Thr Ser Ser Leu Leu 500 505 510Gln Asp Gly
Ile Thr Thr Thr Gln Ala Glu Ile Ser Thr Ser Thr Ile 515 520 525Ser
Thr Ser Pro Thr Ala Gly Ala Ser Leu His His Ala His Asn Phe 530 535
540Ser Phe Leu Gly Ser Phe Asn Met Asp Asn Thr Thr Thr Thr Val
Asp545 550 555 560His Ile Glu Asn Asn Ala Lys Lys Gln Arg Asn Asp
Met His Lys Gly 565 570 575Ser Pro Ser Ser Ile Leu Ser Ser Val Asp
Met Glu Ala Lys Ala Arg 580 585 590Glu Ser Ser Asn Lys Gly Phe Thr
Asn Pro Leu Met Ala Ala Tyr Ala 595 600 605Met Gly Asp Phe Gly Arg
Phe Asp Pro His Asp Gln Gln Met Thr Ala 610 615 620Asn Phe His Gly
Asn Asn Gly Val Ser Leu Thr Leu Gly Leu Pro Pro625 630 635 640Ser
Glu Asn Leu Ala Met Pro Val Ser Gln Gln Asn Tyr Leu Ser Asn 645 650
655Asp Leu Gly Ser Arg Ser Glu Met Gly Ser His Tyr Asn Arg Met Gly
660 665 670Tyr Glu Asn Ile Asp Phe Gln Ser Gly Asn Lys Arg Phe Pro
Thr Gln 675 680 685Leu Leu Pro Asp Phe Val Thr Gly Asn Leu Gly Thr
690 695 70052721PRTArtificialSolanum tuberosum L. 52Ser Leu Ala Asn
Met Thr Asx Glu Arg Ser Met Leu Met Ala Met Tyr1 5 10 15Tyr Gln Gly
Gly Ser Glu Ile Gln Ala Asp Gly Leu Gln Thr Leu Tyr 20 25 30Leu Met
Asn Pro Asn Tyr Ile Gly Tyr Thr Asp Thr His His His His 35 40 45His
Gln His Gln Gln Gln Ser Ala Asn Met Phe Phe Leu Asn Ser Val 50 55
60Ala Ala Gly Asn Phe Pro His Val Ser Leu Pro Leu Gln Ala His Ala65
70 75 80Gln Gly His Leu Val Gly Val Pro Leu Pro Ala Gly Phe Gln Asp
Pro 85 90 95Asn Arg Pro Ser Ile Gln Glu Ile Pro Thr Ser His His Gly
Leu Ile 100 105 110Ser Arg Leu Trp Thr Ser Gly Asp Gln Asn Thr Pro
Arg Gly Gly Gly 115 120 125Gly Gly Gly Glu Gly Asn Gly Ser Gln Ser
His Ile Pro Ser Ser Thr 130 135 140Val Val Ser Pro Asn Ser Gly Ser
Gly Gly Gly Thr Thr Thr Asp Phe145 150 155 160Ala Ser Gln Leu Gly
Phe Gln Arg Pro Gly Leu Val Ser Pro Thr Gln 165 170 175Ala His His
Gln Gly Leu Ser Leu Ser Leu Ser Pro Gln Gln Gln Met 180 185 190Asn
Phe Arg Ser Ser Leu Pro Leu Asp His Arg Asp Ile Ser Thr Thr 195 200
205Asn His Gln Val Gly Ile Leu Ser Ser Ser Pro Leu Pro Ser Pro Gly
210 215 220Thr Asn Thr Asn His Thr Arg Gly Leu Gly Ala Ser Ser Ser
Phe Ser225 230 235 240Ile Ser Asn Gly Met Ile Leu Gly Ser Lys Tyr
Leu Lys Val Ala Gln 245 250 255Asp Leu Leu Asp Glu Val Val Asn Val
Gly Lys Asn Ile Lys Leu Ser 260 265 270Glu Val Gly Ala Lys Glu Lys
His Lys Leu Asp Asn Glu Leu Ile Ser 275 280 285Leu Ala Ser Asp Asp
Val Glu Ser Ser Ser Gln Lys Asn Ser Gly Val 290 295 300Glu Leu Thr
Thr Ala Gln Arg Gln Glu Leu Gln Met Lys Lys Ala Lys305 310 315
320Leu Val Ser Met Leu Asp Glu Val Asp Gln Arg Tyr Arg Gln Tyr His
325 330 335His Gln Met Gln Met Ile Ala Thr Ser Phe Glu Gln Thr Thr
Gly Ile 340 345 350Gly Ser Ser Lys Ser Tyr Thr Gln Leu Ala Leu His
Thr Ile Ser Lys 355 360 365Gln Phe Arg Cys Leu Lys Asp Ala Ile Ser
Gly Gln Ile Lys Asp Thr 370 375 380Ser Lys Thr Leu Gly Glu Glu Glu
Asn Ile Gly Gly Lys Ile Glu Gly385 390 395 400Ser Lys Leu Lys Phe
Val Asp His His Leu Arg Gln Gln Arg Ala Leu 405 410 415Gln Gln Leu
Gly Met Met Gln Thr Asn Ala Trp Arg Pro Gln Arg Gly 420 425 430Leu
Pro Glu Arg Ala Val Ser Val Leu Arg Ala Trp Leu Phe Glu His 435 440
445Phe Leu His Pro Tyr Pro Lys Asp Ser Asp Lys Ile Met Leu Ala Lys
450 455 460Gln Thr Gly Leu Thr Arg Ser Gln Val Ser Asn Trp Phe Ile
Asn Ala465 470 475 480Arg Val Arg Leu Trp Lys Pro Met Val Glu Glu
Met Tyr Met Glu Glu 485 490 495Val Lys Lys Asn Asn Gln Glu Gln Asn
Ile Glu Pro Asn Asn Asn Glu 500 505 510Ile Val Gly Ser Lys Ser Ser
Val Pro Gln Glu Lys Leu Pro Ile Ser 515 520 525Ser Asn Ile Ile His
Asn Ala Ser Pro Asn Asp Ile Ser Thr Ser Thr 530 535 540Ile Ser Thr
Ser Pro Thr Gly Gly Gly Gly Ser Ile Pro Ala Gln Thr545 550 555
560Val Ala Gly Phe Ser Phe Ile Arg Ser Leu Asn Met Glu Asn Ile Asp
565 570 575Asp Gln Arg Asn Asn Lys Lys Ala Arg Asn Glu Met Gln Asn
Cys Ser 580 585 590Thr Ser Thr Ile Leu Ser Met Glu Arg Glu Ile Met
Asn Lys Val Val 595 600 605Gln Asp Glu Thr Ile Lys Ser Glu Lys Phe
Asn Asn Thr Gln Thr Arg 610 615 620Glu Cys Tyr Ser Leu Met Thr Pro
Asn Tyr Thr Met Asp Asp Gln Phe625 630 635 640Gly Thr Arg Phe Asn
Asn Gln Asn His Glu Gln Leu Ala Thr Thr Thr 645 650 655Thr Thr Phe
His Gln Gly Asn Gly His Val Ser Leu Thr Leu Gly Leu 660 665 670Pro
Pro Asn Ser Glu Asn Gln His Asn Tyr Ile Gly Leu Glu Asn His 675 680
685Tyr Asn Gln Pro Thr His His Pro Asn Ile Ser Tyr Glu Asn Ile Asp
690 695 700Phe Gln Ser Gly Lys Arg Tyr Ala Thr Gln Leu Ile Gln Asp
Phe Val705 710 715 720Ser53722PRTArtificialSolanum tuberosum L.
53Ser Leu Ala Asn Met Thr Asx Glu Arg Ser Met Leu Met Ala Ser Tyr1
5 10 15Phe His Gly Asn Ser Glu Ile His Glu Gly Asn Asp Gly Leu Gln
Thr 20 25 30Leu Ile Leu Met Asn Pro Gly Tyr Val Gly Phe Ser Glu Thr
Gln His 35 40 45His His Ala Pro Pro Pro Pro Pro Pro Gly Gly Ser Ser
Ser Asn Ile 50 55 60Val Phe Phe Asn Ser Asn Pro Ile Gly Asn Ser Met
Asn Leu Ser His65 70 75 80Ala Pro Pro Pro Pro Pro Pro Pro Gln Gln
Gln Phe Ile Gly Ile Pro 85 90 95Leu Ala Thr Ala Ala Phe Thr Ala Pro
Ser Gln Asp Ser Gly Asn Asn 100 105 110Asn Asn Asn Glu Ser Ile Ser
Ala Leu His Gly Phe Leu Ala Arg Ser 115 120 125Ser Gln Tyr Gly Phe
Tyr Asn Pro Ala Asn Asp Leu Thr Ala Ala Arg 130 135 140Asp Val Thr
Arg Ala His His His His Gln Gln Pro Arg Ala Phe Thr145 150 155
160Tyr Leu Ser Ser Ser Gln Gln Pro Gly Phe Gly Asn Phe Thr Ala Ala
165 170 175Arg Glu Leu Val Ser Ser Pro Ser Gly Ser Ala Ser Ala Ser
Gly Ile 180 185 190Gln Gln Gln Gln Gln Gln Gln Gln Ser Ile Ser Ser
Val Pro Leu Ser 195 200 205Ser Lys Tyr Met Lys Ala Ala Gln Glu Leu
Leu Asp Glu Val Val Asn 210 215 220Val Gly Lys Ser Met Lys Ser Thr
Asn Ser Thr Asp Val Val Val Asn225 230 235 240Asn Asp Val Lys Lys
Ser Lys Asn Met Gly Asp Met Asp Gly Gln Leu 245 250 255Asp Gly Val
Gly Ala Asp Lys Asp Gly Ala Pro Thr Thr Glu Leu Ser 260
265 270Thr Gly Glu Arg Gln Glu Ile Gln Met Lys Lys Ala Lys Leu Val
Asn 275 280 285Met Leu Asp Glu Val Glu Gln Arg Tyr Arg His Tyr His
His Gln Met 290 295 300Gln Ser Val Ile His Trp Leu Glu Gln Ala Ala
Gly Ile Gly Ser Ala305 310 315 320Lys Thr Tyr Thr Ala Leu Ala Leu
Gln Thr Ile Ser Lys Gln Phe Arg 325 330 335Cys Leu Lys Asp Ala Ile
Ile Gly Gln Ile Arg Ser Ala Ser Gln Thr 340 345 350Leu Gly Glu Glu
Asp Ser Leu Gly Gly Lys Ile Glu Gly Ser Arg Leu 355 360 365Lys Phe
Val Asp Asn Gln Leu Arg Gln Gln Arg Ala Leu Gln Gln Leu 370 375
380Gly Met Ile Gln His Asn Ala Trp Arg Pro Gln Arg Gly Leu Pro
Glu385 390 395 400Arg Ala Val Ser Val Leu Arg Ala Trp Leu Phe Glu
His Phe Leu His 405 410 415Pro Tyr Pro Lys Asp Ser Asp Lys Met Met
Leu Ala Lys Gln Thr Gly 420 425 430Leu Thr Arg Ser Gln Val Ser Asn
Trp Phe Ile Asn Ala Arg Val Arg 435 440 445Leu Trp Lys Pro Met Val
Glu Glu Met Tyr Leu Glu Glu Ile Lys Glu 450 455 460His Glu Gln Asn
Gly Leu Gly Gln Glu Lys Thr Ser Lys Leu Gly Glu465 470 475 480Gln
Asn Glu Asp Ser Thr Thr Ser Arg Ser Ile Ala Thr Gln Asp Lys 485 490
495Ser Pro Gly Ser Asp Ser Gln Asn Lys Ser Phe Val Ser Lys Gln Asp
500 505 510Asn His Leu Pro Gln His Asn Pro Ala Ser Pro Met Pro Asp
Val Gln 515 520 525Arg His Phe His Thr Pro Ile Gly Met Thr Ile Arg
Asn Gln Ser Ala 530 535 540Gly Phe Asn Leu Ile Gly Ser Pro Glu Ile
Glu Ser Ile Asn Ile Thr545 550 555 560Gln Gly Ser Pro Lys Lys Pro
Arg Asn Asn Glu Met Leu His Ser Pro 565 570 575Asn Ser Ile Pro Ser
Ile Asn Met Asp Val Lys Pro Asn Glu Glu Gln 580 585 590Met Ser Met
Lys Phe Gly Asp Asp Arg Gln Asp Arg Asp Gly Phe Ser 595 600 605Leu
Met Gly Gly Pro Met Asn Phe Met Gly Gly Phe Gly Ala Tyr Pro 610 615
620Ile Gly Glu Ile Ala Arg Phe Ser Thr Glu Gln Phe Ser Ala Pro
Tyr625 630 635 640Ser Thr Ser Gly Thr Val Ser Leu Thr Leu Gly Leu
Pro His Asn Glu 645 650 655Asn Leu Ser Met Ser Ala Thr His His Ser
Phe Leu Pro Ile Pro Thr 660 665 670Gln Asn Ile Gln Ile Gly Ser Glu
Pro Asn His Glu Phe Gly Ser Leu 675 680 685Asn Thr Pro Thr Ser Ala
His Ser Thr Ser Ser Val Tyr Glu Thr Phe 690 695 700Asn Ile Gln Asn
Arg Lys Arg Phe Ala Ala Pro Leu Leu Pro Asp Phe705 710 715 720Val
Ala
* * * * *
References